Vista modulators for diagnosis and treatment of cancer

ABSTRACT

The present disclosure relates to compositions and therapeutic methods for activating an immune response in a patient in need thereof. In a preferred embodiment, the subject methods and compositions are able to antagonize the activity of VISTA, a naturally occurring “checkpoint” protein which contributes to immune tolerance, optionally in combination with an antagonist of a second checkpoint pathway such as PD-1. For example, such methods and compositions may be suitable for preventing and treating colon cancer or another cancer. An exemplary VISTA antagonist, specifically, an anti-VISTA antibody, is demonstrated herein to activate an immune response against cancer cells in vitro and in vivo, thereby conferring protective anti-tumor immunity which decreased tumor burden. Additionally, an additive benefit was observed when a VISTA antagonist was used in combination with a second checkpoint protein antagonist, specifically, an antibody against PD-1 ligand (PD-L1).

RELATED APPLICATION DISCLOSURE

This application claims the benefit of U.S. Ser. No. 61/753,682, filedJan. 17, 2013, entitled “VISTA MODULATORS FOR DIAGNOSIS AND TREATMENT OFCANCER”, and also is a continuation-in-part of international applicationno. PCT/US13/58785, filed Sep. 9, 2013, entitled “VISTA MODULATORS FORDIAGNOSIS AND TREATMENT OF CANCER” which claims the benefit of U.S. Ser.No. 61/698,003, filed Sep. 7, 2012 and is a continuation-in-part ofinternational application no. PCT/US2013/047009, filed Jun. 21, 2013,entitled “NOVEL VISTA-IG CONSTRUCTS AND THE USE OF VISTA-IG FORTREATMENT OF AUTOIMMUNE, ALLERGIC AND INFLAMMATORY DISORDERS” whichclaims the benefit of U.S. Provisional Application Ser. No. 61/663,431,filed Jun. 22, 2012, entitled “VISTA-IG FOR TREATMENT OF AUTOIMMUNEDISORDERS AND INFLAMMATORY DISORDERS”, U.S. Provisional Application Ser.No. 61/663,969, filed Jun. 25, 2012, entitled “VISTA-IG FOR TREATMENT OFAUTOIMMUNE DISORDERS AND INFLAMMATORY DISORDERS”, U.S. ProvisionalApplication Ser. No. 61/735,799, filed Dec. 11, 2012, U.S. ProvisionalApplication Ser. No. 61/776,234, filed Mar. 11, 2013, and U.S.Provisional Application Ser. No. 61/807,135, filed Apr. 1, 2013, each ifwhich is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

The sequence listing in the filed named “43260.0701.txt” having a sizeof 112,843 bytes that was created Jul. 7, 2014 is hereby incorporated byreference in its entirety.

GOVERNMENT FUNDING

This invention was made with government support under Grant # AT005382and Grant # AI098007 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD

The present disclosure relates to compositions and therapeutic methodsfor activating an immune response in a patient in need thereof. In apreferred embodiment, the subject methods and compositions are able toantagonize the activity of VISTA, a naturally occurring “checkpoint”protein which contributes to immune tolerance, optionally in combinationwith an antagonist of a second checkpoint pathway such as PD-1. Forexample, such methods and compositions may be suitable for preventingand treating colon cancer or another cancer. An exemplary VISTAantagonist, specifically, an anti-VISTA antibody, is demonstrated hereinto activate an immune response against cancer cells in vitro and invivo, thereby conferring protective anti-tumor immunity which decreasedtumor burden. Additionally, an additive benefit was observed when aVISTA antagonist was used in combination with a second checkpointprotein antagonist, specifically, an antibody against PD-1 ligand(PD-L1).

In another aspect, the disclosure relates to diagnostic methodscomprising measuring the level of expression of VISTA to diagnosedisease mediated by immune tolerance. For example, detection of highlevels of VISTA expression (e.g., VISTA protein or mRNA) in a patientsample may indicate the presence of a cancer. Additionally, thesediagnostic tests may be used to assign a treatment to a patient, forexample by administering a VISTA antagonist based upon the detection ofa high level of VISTA expression in the patient's sample.

BACKGROUND

Immune responses against foreign pathogens and cancer are regulated bymultiple checkpoints, including CTLA-4, PD-L1/PD-1 and B7-H4 pathways.They function as “effector molecules” on multiple immunosuppressivecells, including Tregs, myeloid-derived suppressors (MDSCs) andtolerogenic DCs, to disable tumour-specific T-cell responses.

CTLA-4 is induced on T cells upon activation, and constitutivelyexpressed on Foxp3+CD4+CD25+ natural Tregs (nTreg). CTLA-4 criticallyregulates peripheral tolerance, suppresses T-cell responses, andcontributes to Treg-mediated immune suppression (refs. 6-10). Thecritical role of CTLA-4 in suppressing tumour-specific immunity isdemonstrated when antibody-mediated CTLA-4 blockade in combination witha cellular vaccine induced regression of established poorly immunogenicB16 melanoma (11). Ipilimumab, the human aCTLA-4 mAb, has been approvedfor treating advanced melanoma, although the survival response inmetastatic melanoma is modest (12). It has also undergone early phasetrials for other cancers (13). However, consistent with the severeautoimmune phenotypes in CTLA-4 knockout (KO) mice, aCTLA-4 therapy wasassociated with serious autoimmune toxicity in patients (14).

Programmed Death-1 (PD-1) and its ligand PD-L1 represent another immunecheckpoint pathway (refs. 15, 16). PD-1 KO mice developed autoimmunedisease (refs. 17, 18). In cancer, aberrant PD-L1 expression is seen ontumour cells, which correlates with poorer prognosis in cancer patients(refs. 19, 20). PD-L1/PD-1 axis down-regulates tumour-specific immunityby inducing T-cell apoptosis, anergy, resistance to cytotoxic T-cellmediated lysis, functional exhaustion, and IL10 production (refs.21-23). We and others previously demonstrated that PD-L1 expression onDCs promotes the induction of Foxp3+ adaptive Tregs (aTregs), and PD-L1is a potent inducer of aTregs within the TME (2). Blocking thePD-L1/PD-1 pathway, in conjunction with other immune therapies such asCTLA-4 blockade, inhibits tumour progression (refs. 24-29). MDX-1106,the human aPD-1 mAb has entered clinical trials showing promisinganti-tumour effect, and reduced toxicity compared to Ipilumimab (30).

B7-H4 is a newer member of the B7 inhibitory ligand family (ref. 31-33).B7-H4 expression is detected on many human cancers. In human ovariancancer, B7-H4 expression is induced on tumour associated macrophages(TAM), and its blockade restored tumour-specific T-cell responses andcontributed to tumour regression (34). Human Tregs also conveysuppressive activity to APCs by upregulating B7-H4 expression throughIL10 produced by APCs (35).

In summary, immune-checkpoint blockade improved both endogenous andvaccine-elicited anti-tumour immune responses, yet only produced limitedresponses in clinical trials.

Foxp3+CD4+CD25+ regulatory T cells (Tregs) are critical in maintainingperipheral tolerance under normal physiological conditions, as well assuppressing anti-tumour immune responses in cancer (36-38). In humanovarian cancer, large infiltration of Foxp3+ Tregs is associated withreduced survival (39). Systemic removal of Tregs or attenuation of theirfunctions enhances natural and vaccine-induced antitumor T-cellresponses, resulting in improved therapeutic efficacy (37, 40). Tregsactivated by IDO+ plasmacytoid DCs upregulate B7-H1 expression on targetDCs, and suppress T-cell responses in a PD-L1 dependent manner (41).

Monocytes are precursors for tissue macrophages and monocyte-derived DCs(mo-DC), which play critical roles for both innate and adaptive immunity(42-46). Murine monocytes are identified as CD115+CD11b+F4/80+ (47),consisting of two subsets LY6C+CX3CR1^(int) and LY6C-CX3CR1^(hi) (48,49). The human counterparts are CD14+CD16-CCR2+CX3CR1^(int) andCD14loCD16+CX3CR1^(hi) monocytes respectively. Murine Ly6C+ inflammatorymonocytes (IMC) are recruited to inflammatory sites and differentiate toM1 macrophages and inflammatory mo-DCs, which produce high levels ofTNF/iNOS (Tip DCs) and are critical for microbial clearance43, 50-53. Incontrast, resident LY6C^(neg) monocytes patrol blood vessels in thesteady state, and differentiate into M2-like macrophages duringinfection and inflammation (46).

IMC critically influence the adaptive immune response. In man, TLRinduces the differentiation of monocytes into macrophages and mo-DCs,which are required for optimal T-cell responses (54, 55). In mousemodels, monocyte-derived M1 macrophages and mo-DCs are essential for theinduction of T cell immunity against microbial infection or vaccination,via the production of inflammatory cytokines such as IL-12, and directT-cell priming (56-58).

In tumour-bearing mice and cancer patients, IMCs expand aberrantly andcontribute to the mononuclear subset of myeloid-derived suppressor cells(MDSC) (59-61). MDSCs are collectively marked as CD11b+Gr1+, consistingof the mononuclear (Ly6G+/−LY6C^(−hi)) and the granulocytic(Ly6G+LY6C^(low)) subset (62). MDSCs suppress T cells responses andimpede the efficacy of cancer immunotherapies (60, 62-64). Strategies toeliminate MDSCs, or neutralize their activity, or induce theirdifferentiation have shown efficacy in cancer immunotherapy (60, 63).The majority of tumour-associated DCs are monocyte-derived DCs. They aretypically defective in antigen-presentation, lack costimulatorymolecules, and upregulate inhibitory molecules such as PD-L1 (29, 65,66). As such, these mo-DCs do not effectively prime T-cell responses,resulting in deletional tolerance, or the induction of functionallyinert T cells, and even the expansion and induction of Tregs (40, 60,62, 63, 67, 68). Therapeutic targeting of tumour DCs by PD-L1 blockade,CD40/TLR stimulation, or immunotoxin-mediated depletion significantlyincreased tumour-specific T-cell responses and enhanced survival (29,69-74).

We have recently discovered a novel Immunoglobulin (Ig) family ligand,designated V-domain Immunoglobulin Suppressor of T cell Activation(VISTA) (Genbank: JN602184)75. Key features of VISTA include thefollowing. VISTA bears limited homology to PD-L1, but does not belong tothe B7 family due to its unique structure. VISTA is exclusivelyexpressed within the hematopoietic compartment, with very high levels ofexpression on CD11b^(high) myeloid cells, and lower expression levels onCD4+ and CD8+ T cells, and Tregs. A soluble VISTA-Ig fusion protein orVISTA expressed on APCs, acts as a ligand to suppress CD4+ and CD8+ Tcell proliferation and cytokine production, via an unidentified receptorindependent of PD-1. An anti-VISTA mAb (13F3) reversed VISTA-mediated Tcell suppression in vitro and suppressed tumour growth in multiplemurine tumour models by enhancing the anti-tumour T cell responses.VISTA over-expression on tumour cells impaired protective anti-tumourimmunity in vaccinated hosts. VISTA KO mice develop an inflammatoryphenotype, which points towards a loss of peripheral tolerance. See U.S.Pat. Nos. 8,236,304 and 8,231,872, Published International ApplicationsWO/2011/120013 and WO/2006/116181, U.S. Published Application Nos.2008/0287358, 2011/0027278, and 2012/0195894, and U.S. ProvisionalPatent Application Ser Nos. 60/674,567, filed Apr. 25, 2005, 61/663,431,filed Jun. 22, 2012, Ser. No. 61/663,969, filed Jun. 25, 2012,61/390,434, filed Oct. 6, 2010, 61/436,379, filed Jan. 26, 2011, and61/449,882, filed Mar. 7, 2011, each of which is hereby incorporated byreference in its entirety.

We therefore hypothesize that VISTA is a novel immune checkpoint proteinligand that critically regulates immune responses, and VISTA blockadewill reverse the suppressive character of the tumour microenvironment(TME) and lead to the development of protective anti-tumour immunity.

The immune system is tightly controlled by co-stimulatory andco-inhibitory ligands and receptors. These molecules provide not only asecond signal for T cell activation but also a balanced network ofpositive and negative signals to maximize immune responses againstinfection while limiting immunity to self.

Induction of an immune response requires T cell expansion,differentiation, contraction and establishment of T cell memory. T cellsmust encounter antigen presenting cells (APCs) and communicate via Tcell receptor (TCR)/major histocompatibility complex (MHC) interactionson APCs. Once the TCR/MHC interaction is established, other sets ofreceptor-ligand contacts between the T cell and the APC are required,i.e. co-stimulation via CD154/CD40 and CD28/B7.1-B7.2. The synergybetween these contacts results in a productive immune response capableof clearing pathogens and tumors, and may be capable of inducingautoimmunity.

Another level of control has been identified, namely regulatory T cells(T_(reg)). This specific subset of T cells is generated in the thymus,delivered into the periphery, and is capable of constant and induciblecontrol of T cells responses. Sakaguchi (2000) Cell 101(5):455-8;Shevach (2000) Annu. Rev. Immunol 18:423-49; Bluestone and Abbas (2003)Nat. Rev. Immunol. 3(3):253-7. T_(reg) are represented by a CD4+CD25+phenotype and also express high levels of cytotoxic Tlymphocyte-associated antigen-4 (CTLA-4), OX-40, 4-1BB and theglucocorticoid inducible TNF receptor-associated protein (GITR). McHugh,et al. (2002) Immunity 16(2):311-23; Shimizu, et al. (2002) Nat. Immun.3(2):135-42. Elimination of T_(reg) cells by 5 day neonatal thymectomyor antibody depletion using anti-CD25, results in the induction ofautoimmune pathology and exacerbation of T cells responses to foreignand self-antigens, including heightened anti-tumor responses. Sakaguchi,et al. (1985) J. Exp. Med. 161(1):72-87; Sakaguchi, et al. (1995) J.Immunol. 155(3):1151-64; Jones, et al. (2002) Cancer Immun. 2:1. Inaddition, T_(reg) have also been involved in the induction andmaintenance of transplantation tolerance, since depletion of T_(reg)with anti-CD25 monoclonal antibodies results in ablation oftransplantation tolerance and rapid graft rejection. Jarvinen, et al.(2003) Transplantation 76:1375-9. Among the receptors expressed byT_(reg) GITR seems to be an important component since ligation of GITRon the surface of Treg with an agonistic monoclonal antibody results inrapid termination of T_(reg) activity, resulting in autoimmune pathologyand ablation of transplantation tolerance.

Costimulatory and co-inhibitory ligands and receptors not only provide a“second signal” for T cell activation, but also a balanced network ofpositive and negative signal to maximize immune responses againstinfection while limiting immunity to self. The best characterizedcostimulatory ligands are B7.1 and B7.2, which are expressed byprofessional APCs, and whose receptors are CD28 and CTLA-4. Greenwald,et al. (2005) Annu Rev Immunol 23, 515-548; Sharpe and Freeman (2002)Nat Rev Immunol 2, 116-126. CD28 is expressed by naïve and activated Tcells and is critical for optimal T cell activation. In contrast, CTLA-4is induced upon T cell activation and inhibits T cell activation bybinding to B7.1/B7.2, thus impairing CD28-mediated costimulation. CTLA-4also transduces negative signaling through its cytoplasmic ITIM motif.Teft, et al. (2006). Annu Rev Immunol 24, 65-97. B7.1/B7.2 KO mice areimpaired in adaptive immune response (Borriello, et al. (1997) Immunity6, 303-313; Freeman, et al. (1993) Science 262, 907-909), whereas CTLA-4KO mice can not adequately control inflammation and develop systemicautoimmune diseases. Chambers, et al. (1997) Immunity 7, 885-895; Tivol,et al. (1995) Immunity 3, 541-547; Waterhouse, et al. (1995) Science270, 985-988. The B7 family ligands have expanded to includecostimulatory B7-H2 (ICOS Ligand) and B7-H3, as well as co-inhibitoryB7-H1 (PD-L1), B7-DC (PD-L2), B7-H4 (B7S1 or B7x), and B7-H6. SeeBrandt, et al. (2009) J Exp Med 206, 1495-1503; Greenwald, et al. (2005)Annu Rev Immunol 23: 515-548.

Inducible costimulatory (ICOS) molecule is expressed on activated Tcells and binds to B7-H2. See Yoshinaga, et al. (1999) Nature 402,827-832. ICOS is important for T cell activation, differentiation andfunction, as well as essential for T-helper-cell-induced B cellactivation, Ig class switching, and germinal center (GC) formation.Dong, et al. (2001) Nature 409, 97-101; Tafuri, et al. (2001) Nature409, 105-109; Yoshinaga, et al. (1999) Nature 402, 827-832. ProgrammedDeath 1 (PD-1) on the other hand, negatively regulates T cell responses.PD-1 KO mice develop lupus-like autoimmune disease, or autoimmunedilated cardiomyopathy depending upon the genetic background. Nishimura,et al. (1999) Immunity 11, 141-151. Nishimura, et al. (2001) Science291: 319-322. The autoimmunity most likely results from the loss ofsignaling by both ligands PD-L1 and PD-L2. Recently, CD80 was identifiedas a second receptor for PD-L1 that transduces inhibitory signals into Tcells. Butte, et al. (2007) Immunity 27: 111-122. The receptor for B7-H3and B7-H4 still remain unknown.

The best characterized co-stimulatory ligands are B7.1 and B7.2, whichbelong to the Ig superfamily and are expressed on professional APCs andwhose receptors are CD28 and CTLA-4. Greenwald, et al. (2005) Annu Rev.Immunol. 23: 515-548. CD28 is expressed by naive and activated T cellsand is critical for optimal T cell activation. In contrast, CTLA-4 isinduced upon T cell activation and inhibits T cell activation by bindingto B7.1/B7.2, impairing CD28-mediated co-stimulation. B7.1 and B7.2 KOmice are impaired in adaptive immune response (Borriello, et al. (1997)Immunity 6: 303-313), whereas CTLA-4 KO mice cannot adequately controlinflammation and develop systemic autoimmune diseases. Tivol, et al.(1995) Immunity 3: 541-547; Waterhouse, et al. (1995) Science 270:985-988; Chambers, et al. (1997) Immunity 7: 885-895.

The B7 family ligands have expanded to include co-stimulatory B7-H2(inducible T cell co-stimulator [ICOS] ligand) and B7-H3, as well asco-inhibitory B7-H1 (PD-L1), B7-DC (PD-L2), B7-H4 (B7S1 or B7x), andB7-H6. Greenwald, et al. (2005) Annu Rev. Immunol. 23: 515-548; Brandt,et al. (2009) J. Exp. Med. 206: 1495-1503. Accordingly, additional CD28family receptors have been identified. ICOS is expressed on activated Tcells and binds to B7-H2. ICOS is a positive coregulator, which isimportant for T cell activation, differentiation, and function.Yoshinaga, et al. (1999) Nature 402: 827-832; Dong, et al. (2001) J.Mol. Med. 81: 281-287. In contrast, PD-1 (programmed death 1) negativelyregulates T cell responses. PD-1 KO mice developed lupus-like autoimmunedisease or autoimmune dilated cardiomyopathy. Nishimura, et al. (1999)Immunity 11: 141-151; Nishimura, et al. (2001) Science 291: 319-322. Theautoimmunity most likely results from the loss of signaling by bothligands PD-L1 and PD-L2. Recently, CD80 was identified as a secondreceptor for PD-L1 that transduces inhibitory signals into T cells.Butte, et al. (2007) Immunity 27: 111-122.

The two inhibitory B7 family ligands, PD-L1 and PD-L2, have distinctexpression patterns. PD-L2 is inducibly expressed on DCs andmacrophages, whereas PD-L1 is broadly expressed on both hematopoieticcells and nonhematopoietic cell types. Okazaki & Honjo (2006) TrendsImmunol 27(4): 195-201; Keir, et al. (2008) Ann Rev Immunol. 26:677-704. Consistent with the immune-suppressive role of PD-1 receptor, astudy using PD-L1^(−/−) and PD-L2^(−/−) mice has shown that both ligandshave overlapping roles in inhibiting T cell proliferation and cytokineproduction. Keir, et al. (2006) J Immunol. 175(11): 7372-9. PD-L1deficiency enhances disease progression in both the nonobese diabeticmodel of autoimmune diabetes and the mouse model of multiple sclerosis(experimental autoimmune encephalomyelitis [EAE]). Ansari, et al. (2003)J. Exp. Med. 198: 63-69; Salama, et al. (2003) J. Exp. Med. 198: 71-78;Latchman, et al. (2004) Proc. Natl. Acad. Sci. USA. 101: 10691-10696.PD-L1^(−/−) T cells produce elevated levels of the proinflammatorycytokines in both disease models. In addition, BM chimera experimentshave demonstrated that the tissue expression of PD-L1 (i.e., withinpancreas) uniquely contributes to its capacity of regionally controllinginflammation. Keir, et al. (2006) J. Exp. Med. 203: 883-895; Keir, etal. (2007) J. Immunol. 179: 5064-5070; Grabie, et al. (2007) Circulation116: 2062-2071. PD-L1 is also highly expressed on placentalsyncytiotrophoblasts, which critically control the maternal immuneresponses to allogeneic fetus. Guleria, et al. (2005) J. Exp. Med. 202:231-237.

Consistent with its immune-suppressive role, PD-L1 potently suppressesantitumor immune responses and helps tumors evade immune surveillance.PD-L1 can induce apoptosis of infiltrating cytotoxic CD8⁺ T cells, whichexpress a high level of PD-1. Dong, et al. (2002) Nat. Med. 8: 793-800;Dong and Chen (2003) J. Mol. Med. 81: 281-287. Blocking the PD-L1-PD-1signaling pathway, in conjunction with other immune therapies, preventstumor progression by enhancing antitumor CTL activity and cytokineproduction. Iwai, et al. (2002) Proc. Natl. Acad. Sci. USA 99:12293-12297; Blank, et al. (2004) Cancer Res. 64: 1140-1145; Blank, etal. (2005) Cancer Immunol. Immunother. 54: 307-314; Geng, et al. (2006)Int. J. Cancer 118: 2657-2664. PD-L1 expression on DCs promotes theinduction of adaptive Foxp3⁺CD4⁺ regulatory T cells (T_(reg) cells), andPD-L1 is a potent inducer of aT_(reg) cells within the tumormicroenvironment. Wang, et al. (2008) Proc Natl. Acad. Sci. USA 105:9331-9336. Recent advances in targeting B7 family regulatory moleculesshow promise in treating immune-related diseases such as autoimmunityand cancer. Keir, et al. (2008) Annu. Rev. Immunol. 26: 677-704; Zou andChen (2008) Nat. Rev. Immunol 8: 467-477.

Autoimmune Disease

An autoimmune disorder is a condition that occurs when the immune systemmistakenly attacks and destroys healthy body tissue. There are more than80 different types of autoimmune disorders. Normally the immune system'swhite blood cells help protect the body from harmful substances, calledantigens. Examples of antigens include bacteria, viruses, toxins, cancercells, and blood or tissues from another person or species. The immunesystem produces antibodies that destroy these harmful substances.However, in patients with an autoimmune disorder, the immune system cannot distinguish between self and non-self (e.g., healthy tissue andforeign antigens). The result is an immune response that destroys normalbody tissues. This response is a hypersensitivity reaction similar tothe response in allergic conditions. In allergies, the immune systemreacts to an outside substance that it normally would ignore. Withautoimmune disorders, the immune system reacts to normal body tissuesthat it would normally ignore, the cause of which is unknown.

An autoimmune disorder may result in the destruction of one or moretypes of body tissue, abnormal growth of an organ, and changes in organfunction and may affect one or more organ or tissue types. Organs andtissues commonly affected by autoimmune disorders include blood vessels,connective tissues, endocrine glands (e.g., thyroid or pancreas),joints, muscles, red blood cells, and skin. A person may have more thanone autoimmune disorder at the same time.

Symptoms of an autoimmune disease vary based on the disease and locationof the abnormal immune response. Common symptoms that often occur withautoimmune diseases include fatigue, fever, and a general ill-feeling(malaise). Tests that may be done to diagnose an autoimmune disorder mayinclude: antinuclear antibody tests, autoantibody tests, CBC, C-reactiveprotein (CRP), and erythrocyte sedimentation rate (ESR).

Medicines are often prescribed to control or reduce the immune system'sresponse. They are often called immunosuppressive medicines. Suchmedicines may include corticosteroids (such as prednisone) andnonsteroid drugs such as azathioprine, cyclophosphamide, mycophenolate,sirolimus, or tacrolimus.

Complications are common and depend on the disease. Side effects ofmedications used to suppress the immune system can be severe, such asinfections that can be hard to control. “Autoimmune disorders.”MedlinePlus—U.S. National Library of Medicine (Apr. 19, 2012).

Inflammatory Conditions

Inflammation is part of the complex biological response of vasculartissues to harmful stimuli, such as pathogens, damaged cells, orirritants. Inflammation is a protective attempt by the organism toremove the injurious stimuli and to initiate the healing process.Without inflammation, wounds and infections would never heal. Similarly,progressive destruction of the tissue would compromise the survival ofthe organism. However, chronic inflammation can also lead to a host ofdiseases, such as hay fever, periodontitis, atherosclerosis, rheumatoidarthritis, and even cancer (e.g., gallbladder carcinoma).

Inflammation can be classified as either acute or chronic. Acuteinflammation is the initial response of the body to harmful stimuli andis achieved by the increased movement of plasma and leukocytes(especially granulocytes) from the blood into the injured tissues. Acascade of biochemical events propagates and matures the inflammatoryresponse, involving the local vascular system, the immune system, andvarious cells within the injured tissue. Prolonged inflammation, knownas chronic inflammation, leads to a progressive shift in the type ofcells present at the site of inflammation and is characterized bysimultaneous destruction and healing of the tissue from the inflammatoryprocess. Kindt, et al. (2006) Kuby Immunology [6^(th) Ed.]

T-cells are involved in the promulgation of inflammation.Differentiation of naïve T cells leads to the generation of T-cellsubsets, each possessing distinct cytokine expression profiles forserving different immune functions. Through the activation of separatesignaling pathways, this process results in both differentiated helper T(Th) cells, termed Th1, Th2 and Th17, and induced regulatory T cells,which suppress Th cells. These different cells are important forcombating infectious diseases and cancers; however, when aberrant, theycan be responsible for chronic inflammatory diseases. One such diseaseis inflammatory bowel disease (IBD), in which each T-cell subset canhave a role in disease. Zenewicz, et al. (2009) Trends in MolecularMedicine 15(5): 199-207. Therefore, T cells are involved in bothautoimmune disorders and inflammatory conditions and there is a need inthe art for a novel molecule that can modulate the activity of T cellsfor the treatment of autoimmune disorders and inflammatory conditions.

SUMMARY

Cancer immunotherapies that target immune checkpoint proteins such asCTLA-4 and PD-1 have shown promising outcomes in clinical trials. Thisis especially promising considering the poor prognosis and treatmentoptions for the patients involved. However, the overall response ratehas been disappointingly low, with 6-21% patients in various ipilimumab(aCTLA-4) trials having objective responses3-5. Therefore, identifyingnovel checkpoint proteins that play a non-redundant role and synergizewith the known checkpoint pathways is critically needed. As a novelimmune checkpoint pathway, VISTA provides a new target for immuneintervention in cancer. VISTA blockade reverses the suppressivecharacter of the TME, and leads to the development of protectiveantitumour immunity. The results described herein help show that VISTAblockade is an effective therapeutic strategy for targeting prominentimmunosuppressive cells, including Tregs and MDSCs in cancer such ascolorectal cancer (CRC).

In one aspect, the present disclosure provides a new paradigm in which anovel immune checkpoint pathway, VISTA, critically controls theanti-tumour immune responses. This paradigm builds a foundation fordesigning novel therapeutic strategies that target the VISTA pathway.The collaborative interaction between VISTA and another immunecheckpoint pathway PD-L1/PD-1 argues against “redundancy”, andemphasizes the necessity to target all of the immunosuppressive pathwaysfor maximal impact. Base thereon, the application further provides novelcombinatorial strategies and change the current regimes of targeting asingle pathway in cancer immunotherapy. Moreover, the study of the roleof VISTA during natural tumourigenesis will generate more clinicallyrelevant information, and guide the development of better therapeuticstrategies.

The invention provides an isolated VISTA fusion protein comprising apolypeptide with at least about 90% sequence identity to theextracellular domain of the polypeptide sequence of SEQ ID NO: 2, 4, 5,16-25, 36, or 37 and an immunoglobulin (Ig) protein.

In one embodiment, the polypeptide may have at least about 95% sequenceidentity to the polypeptide sequence of SEQ ID NO: 2, 4, 5, 16-25, 36,or 37.

In one embodiment, the Ig protein may be IgG, IgG1, IgG2, IgG2a, IgM,IgE, or IgA. In one embodiment, the Ig protein may be the constant andhinge region of human IgG1.

In one embodiment, the extracellular domain of VISTA comprises aminoacid residues 32-190 or the extracellular IgV domain of VISTA maycomprise amino acids 16-194. In one embodiment, the fusion proteincomprises at least two copies of a VISTA protein and IgG1, or IgG2a.

In one embodiment, the fusion protein comprises at least two copies of aVISTA protein and IgG1 Fc or non-FcR-binding IgG1. In one embodiment,the fusion protein comprises at least four copies of a VISTA protein andIgG1 or IgG2a. In one embodiment, the fusion protein comprises at leastfour copies of a VISTA protein and IgG1 Fc or non-FcR-binding IgG1.

In one embodiment, an isolated multimeric VISTA protein may comprise atleast two copies of a polypeptide with at least about 90% sequenceidentical to the extracellular domain may comprise the polypeptidesequence of SEQ ID NO: 2, 4, or 25. In another embodiment, thepolypeptide may have at least about 95% sequence identity to thepolypeptide sequence of SEQ ID NO: 2, 4, 5, 16-25, 36, or 37. In anotherembodiment, the polypeptide may have at least about 90% sequenceidentity to a fragment of the extracellular domain of said VISTApolypeptide which may be at least 50 amino acids long.

In another embodiment, the fragment of the extracellular domain of saidVISTA polypeptide may be at least about 75 amino acids long. In anotherembodiment, the fragment of the extracellular domain of said VISTApolypeptide may be at least about 100 amino acids long. In anotherembodiment, the fragment of the extracellular domain of said VISTApolypeptide may be at least about 125 amino acids long.

In another embodiment, the multimeric VISTA protein comprises at leastthree copies of said extracellular domain or fragment thereof. Inanother embodiment, the multimeric VISTA protein comprises at least fourcopies of said extracellular domain or fragment thereof. In anotherembodiment, the multimeric VISTA protein at least five copies of saidextracellular domain or fragment thereof. In another embodiment, themultimeric VISTA protein at least six copies of said extracellulardomain or fragment thereof.

In another embodiment, the extracellular domain or fragment may beattached to the N-terminus of an oligomerization domain. In anotherembodiment, the oligomerization domain may be GCN4, COMP, SNARE, CMP,MAT, LLR containing 1 NLRC, NOD2 nucleotide-binding NLRC2, LRRcontaining 1 NLRC NOD2 nucleotide-binding NLRC2, or PSORAS1.

In one embodiment, a composition may comprise the VISTA fusion protein.In one embodiment, a composition may comprise the multimeric VISTAprotein. In another embodiment, the composition may be a pharmaceuticalcomposition. In another embodiment, the pharmaceutical composition maycomprise a pharmaceutically acceptable carrier, excipient, adjuvant, orsolution. In another embodiment, the composition may further comprise atleast one other immunosuppressive agent. In another embodiment, theimmunosuppressive agent may be PD-1, PD-L1, PD-L2, CTLA4, ICOS proteins,or antibodies specific to any of the foregoing.

In one embodiment, a method of treating or preventing inflammation in asubject in need thereof may comprise administering an effective amountof a VISTA fusion protein, optionally a VISTA-Ig, or a multimeric VISTAprotein.

In one embodiment, a composition for treating or preventing inflammationmay comprise administering an effective amount of a VISTA fusionprotein, optionally a VISTA-Ig, or a multimeric VISTA protein.

In another embodiment, the use of an effective amount of a VISTA fusionprotein, optionally a VISTA-Ig, or a multimeric VISTA protein for themanufacture of a medicament for treating inflammation.

In another embodiment, the subject may have inflammatory condition.

In another embodiment, the inflammatory condition may be AcidReflux/Heartburn, Acne, Acne Vulgaris, Allergies and Sensitivities,Alzheimer's Disease, Asthma, Atherosclerosis and Vascular OcclusiveDisease, optionally Atherosclerosis, Ischaemic Heart Disease, MyocardialInfarction, Stroke, Peripheral Vascular Disease, or Vascular StentRestenosis, Autoimmune Diseases, Bronchitis, Cancer, Carditis,Cataracts, Celiac Disease, Chronic Pain, Chronic Prostatitis, Cirrhosis,Colitis, Connective Tissue Diseases, optionally Systemic LupusErythematosus, Systemic Sclerosis, Polymyositis, Dermatomyositis, orSjogren's Syndrome, Corneal Disease, Crohn's Disease, CrystalArthropathies, optionally Gout, Pseudogout, Calcium PyrophosphateDeposition Disease, Dementia, Dermatitis, Diabetes, Dry Eyes, Eczema,Edema, Emphysema, Fibromyalgia, Gastroenteritis, Gingivitis,Glomerulonephritis, Heart Disease, Hepatitis, High Blood Pressure,Hypersensitivities, Inflammatory Bowel Diseases, Inflammatory Conditionsincluding Consequences of Trauma or Ischaemia, Insulin Resistance,Interstitial Cystitis, Iridocyclitis, Iritis, Joint Pain, Arthritis,Rheumatoid Arthritis, Lyme Disease, Metabolic Syndrome (Syndrome X),Multiple Sclerosis, Myositis, Nephritis, Obesity, Ocular Diseasesincluding Uveitis, Osteopenia, Osteoporosis, Parkinson's Disease, PelvicInflammatory Disease, Periodontal Disease, Polyarteritis,Polychondritis, Polymyalgia Rheumatica, Psoriasis, Reperfusion Injury,Rheumatic Arthritis, Rheumatic Diseases, optionally RheumatoidArthritis, Osteoarthritis, or Psoriatic Arthritis, Rheumatoid Arthritis,Sarcoidosis, Scleroderma, Sinusitis, Sjögren's Syndrome, Spastic Colon,Spondyloarthropathies, optionally Ankylosing Spondylitis, ReactiveArthritis, or Reiter's Syndrome, Systemic Candidiasis, Tendonitis,Transplant Rejection, UTI's, Vaginitis, Vascular Diseases includingAtherosclerotic Vascular Disease, Vasculitides, optionally PolyarteritisNodosa, Wegener's Granulomatosis, Churg-Strauss Syndrome, or vasculitis.

In one embodiment, a method of treating an autoimmune disease maycomprise administering an effective amount of a VISTA fusion protein,optionally a VISTA-Ig, or a multimeric VISTA protein.

In one embodiment, a composition for treating an autoimmune disease maycomprise administering an effective amount of a VISTA fusion protein,optionally a VISTA-Ig, or a multimeric VISTA protein.

In another embodiment, the use of an effective amount of a VISTA fusionprotein, optionally a VISTA-Ig, or a multimeric VISTA protein for themanufacture of a medicament of the treatment of an autoimmune disease.

In another embodiment, the autoimmune disease may be a cell mediatedautoimmune disease.

In another embodiment, the cell mediate autoimmune disease may bemultiple sclerosis, diabetes type I, oophoritis, or thyroiditis.

In another embodiment, the autoimmune disease may be acquired immunedeficiency syndrome (AIDS), acquired spenic atrophy, acute anterioruveitis, Acute Disseminated Encephalomyelitis (ADEM), acute goutyarthritis, acute necrotizing hemorrhagic leukoencephalitis, acute orchronic sinusitis, acute purulent meningitis (or other central nervoussystem inflammatory disorders), acute serious inflammation, Addison'sdisease, adrenalitis, adult onset diabetes mellitus (Type II diabetes),adult-onset idiopathic hypoparathyroidism (AOIH), Agammaglobulinemia,agranulocytosis, vasculitides, including vasculitis, optionally, largevessel vasculitis, optionally, polymyalgia rheumatica and giant cell(Takayasu's) arthritis, allergic conditions, allergic contactdermatitis, allergic dermatitis, allergic granulomatous angiitis,allergic hypersensitivity disorders, allergic neuritis, allergicreaction, alopecia greata, alopecia totalis, Alport's syndrome,alveolitis, optionally allergic alveolitis or fibrosing alveolitis,Alzheimer's disease, amyloidosis, amylotrophic lateral sclerosis (ALS;Lou Gehrig's disease), an eosinophil-related disorder, optionallyeosinophilia, anaphylaxis, ankylosing spondylitis, antgiectasis,antibody-mediated nephritis, Anti-GBM/Anti-TBM nephritis,antigen-antibody complex-mediated diseases, antiglomerular basementmembrane disease, anti-phospholipid antibody syndrome, antiphospholipidsyndrome (APS), aphthae, aphthous stomatitis, aplastic anemia,arrhythmia, arteriosclerosis, arteriosclerotic disorders, arthritis,optionally rheumatoid arthritis such as acute arthritis, or chronicrheumatoid arthritis, arthritis chronica progrediente, arthritisdeformans, ascariasis, aspergilloma, granulomas containing eosinophils,aspergillosis, aspermiogenese, asthma, optionally asthma bronchiale,bronchial asthma, or auto-immune asthma, ataxia telangiectasia, ataxicsclerosis, atherosclerosis, autism, autoimmune angioedema, autoimmuneaplastic anemia, autoimmune atrophic gastritis, autoimmune diabetes,autoimmune disease of the testis and ovary including autoimmune orchitisand oophoritis, autoimmune disorders associated with collagen disease,autoimmune dysautonomia, autoimmune ear disease, optionally autoimmuneinner ear disease (AGED), autoimmune endocrine diseases includingthyroiditis such as autoimmune thyroiditis, autoimmune enteropathysyndrome, autoimmune gonadal failure, autoimmune hearing loss,autoimmune hemolysis, Autoimmune hepatitis, autoimmune hepatologicaldisorder, autoimmune hyperlipidemia, autoimmune immunodeficiency,autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmuneneutropenia, autoimmune pancreatitis, autoimmune polyendocrinopathies,autoimmune polyglandular syndrome type I, autoimmune retinopathy,autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease,autoimmune urticaria, autoimmune-mediated gastrointestinal diseases,Axonal & neuronal neuropathies, Balo disease, Behcet's disease, benignfamilial and ischemia-reperfusion injury, benign lymphocytic angiitis,Berger's disease (IgA nephropathy), bird-fancier's lung, blindness,Boeck's disease, bronchiolitis obliterans (non-transplant) vs NSIP,bronchitis, bronchopneumonic aspergillosis, Bruton's syndrome, bullouspemphigoid, Caplan's syndrome, Cardiomyopathy, cardiovascular ischemia,Castleman's syndrome, Celiac disease, celiac sprue (gluten enteropathy),cerebellar degeneration, cerebral ischemia, and disease accompanyingvascularization, Chagas disease, channelopathies, optionally epilepsy,channelopathies of the CNS, chorioretinitis, choroiditis, an autoimmunehematological disorder, chronic active hepatitis or autoimmune chronicactive hepatitis, chronic contact dermatitis, chronic eosinophilicpneumonia, chronic fatigue syndrome, chronic hepatitis, chronichypersensitivity pneumonitis, chronic inflammatory arthritis, Chronicinflammatory demyelinating polyneuropathy (CIDP), chronic intractableinflammation, chronic mucocutaneous candidiasis, chronic neuropathy,optionally IgM polyneuropathies or IgM-mediated neuropathy, chronicobstructive airway disease, chronic pulmonary inflammatory disease,Chronic recurrent multifocal ostomyelitis (CRMO), chronic thyroiditis(Hashimoto's thyroiditis) or subacute thyroiditis, Churg-Strausssyndrome, cicatricial pemphigoid/benign mucosal pemphigoid, CNSinflammatory disorders, CNS vasculitis, Coeliac disease, Coganssyndrome, cold agglutinin disease, colitis polyposa, colitis such asulcerative colitis, colitis ulcerosa, collagenous colitis, conditionsinvolving infiltration of T cells and chronic inflammatory responses,congenital heart block, congenital rubella infection, Coombs positiveanemia, coronary artery disease, Coxsackie myocarditis, CREST syndrome(calcinosis, Raynaud's phenomenon), Crohn's disease, cryoglobulinemia,Cushing's syndrome, cyclitis, optionally chronic cyclitis, heterochroniccyclitis, iridocyclitis, or Fuch's cyclitis, cystic fibrosis,cytokine-induced toxicity, deafness, degenerative arthritis,demyelinating diseases, optionally autoimmune demyelinating diseases,demyelinating neuropathies, dengue, dermatitis herpetiformis and atopicdermatitis, dermatitis including contact dermatitis, dermatomyositis,dermatoses with acute inflammatory components, Devic's disease(neuromyelitis optica), diabetic large-artery disorder, diabeticnephropathy, diabetic retinopathy, Diamond Blackfan anemia, diffuseinterstitial pulmonary fibrosis, dilated cardiomyopathy, discoid lupus,diseases involving leukocyte diapedesis, Dressler's syndrome,Dupuytren's contracture, echovirus infection, eczema including allergicor atopic eczema, encephalitis such as Rasmussen's encephalitis andlimbic and/or brainstem encephalitis, encephalomyelitis, optionallyallergic encephalomyelitis or encephalomyelitis allergica andexperimental allergic encephalomyelitis (EAE), endarterial hyperplasia,endocarditis, endocrine ophthamopathy, endometriosis. endomyocardialfibrosis, endophthalmia phacoanaphylactica, endophthalmitis, enteritisallergica, eosinophilia-myalgia syndrome, eosinophilic faciitis,epidemic keratoconjunctivitis, epidermolisis bullosa acquisita (EBA),episclera, episcleritis, Epstein-Barr virus infection, erythema elevatumet diutinum, erythema multiforme, erythema nodosum leprosum, erythemanodosum, erythroblastosis fetalis, esophageal dysmotility, Essentialmixed cryoglobulinemia, ethmoid, Evan's syndrome, Experimental AllergicEncephalomyelitis (EAE), Factor VIII deficiency, farmer's lung, febrisrheumatica, Felty's syndrome, fibromyalgia, fibrosing alveolitis,flariasis, focal segmental glomerulosclerosis (FSGS), food poisoning,frontal, gastric atrophy, giant cell arthritis (temporal arthritis),giant cell hepatitis, giant cell polymyalgia, glomerulonephritides,glomerulonephritis (GN) with and without nephrotic syndrome such aschronic or acute glomerulonephritis (e.g., primary GN), Goodpasture'ssyndrome, gouty arthritis, granulocyte transfusion-associated syndromes,granulomatosis including lymphomatoid granulomatosis, granulomatosiswith polyangiitis (GPA), granulomatous uveitis, Grave's disease,Guillain-Barre syndrome, gutatte psoriasis, haemoglobinuriaparoxysmatica, Hamman-Rich's disease, Hashimoto's disease, Hashimoto'sencephalitis, Hashimoto's thyroiditis, hemochromatosis, hemolytic anemiaor immune hemolytic anemia including autoimmune hemolytic anemia (AIHA),hemolytic anemia, hemophilia A, Henoch-Schonlein purpura, Herpesgestationis, human immunodeficiency virus (HIV) infection, hyperalgesia,hypogammaglobulinemia, hypogonadism, hypoparathyroidism, idiopathicdiabetes insipidus, idiopathic facial paralysis, idiopathichypothyroidism, idiopathic IgA nephropathy, idiopathic membranous GN oridiopathic membranous nephropathy, idiopathic nephritic syndrome,idiopathic pulmonary fibrosis, idiopathic sprue, Idiopathicthrombocytopenic purpura (ITP), IgA nephropathy, IgE-mediated diseases,optionally anaphylaxis and allergic or atopic rhinitis, IgG4-relatedsclerosing disease, ileitis regionalis, immune complex nephritis, immuneresponses associated with acute and delayed hypersensitivity mediated bycytokines and T-lymphocytes, immune-mediated GN, immunoregulatorylipoproteins, including adult or acute respiratory distress syndrome(ARDS), Inclusion body myositis, infectious arthritis, infertility dueto antispermatozoan antibodies, inflammation of all or part of the uvea,inflammatory bowel disease (IBD) inflammatory hyperproliferative skindiseases, inflammatory myopathy, insulin-dependent diabetes (type1),insulitis, Interstitial cystitis, interstitial lung disease,interstitial lung fibrosis, iritis, ischemic re-perfusion disorder,joint inflammation, Juvenile arthritis, juvenile dermatomyositis,juvenile diabetes, juvenile onset (Type I) diabetes mellitus, includingpediatric insulin-dependent diabetes mellitus (IDDM), juvenile-onsetrheumatoid arthritis, Kawasaki syndrome, keratoconjunctivitis sicca,kypanosomiasis, Lambert-Eaton syndrome, leishmaniasis, leprosy,leucopenia, leukocyte adhesion deficiency, Leukocytoclastic vasculitis,leukopenia, lichen planus, lichen sclerosus, ligneous conjunctivitis,linear IgA dermatosis, Linear IgA disease (LAD), Loffler's syndrome,lupoid hepatitis, lupus (including nephritis, cerebritis, pediatric,non-renal, extra-renal, discoid, alopecia), Lupus (SLE), lupuserythematosus disseminatus, Lyme arthritis, Lyme disease, lymphoidinterstitial pneumonitis, malaria, male and female autoimmuneinfertility, maxillary, medium vessel vasculitis (including Kawasaki'sdisease and polyarteritis nodosa), membrano- or membranous proliferativeGN (MPGN), including Type I and Type II, and rapidly progressive GN,membranous GN (membranous nephropathy), Meniere's disease, meningitis,microscopic colitis, microscopic polyangiitis, migraine, minimal changenephropathy, Mixed connective tissue disease (MCTD), mononucleosisinfectiosa, Mooren's ulcer, Mucha-Habermann disease, multifocal motorneuropathy, multiple endocrine failure, multiple organ injury syndromesuch as those secondary to septicemia, trauma or hemorrhage, multipleorgan injury syndrome, multiple sclerosis (MS) such as spino-optical MS,multiple sclerosis, mumps, muscular disorders, myasthenia gravis such asthymoma-associated myasthenia gravis, myasthenia gravis, myocarditis,myositis, narcolepsy, necrotizing enterocolitis, and transmural colitis,and autoimmune inflammatory bowel disease, necrotizing, cutaneous, orhypersensitivity vasculitis, neonatal lupus syndrome (NLE), nephrosis,nephrotic syndrome, neurological disease, neuromyelitis optica(Devic's), neuromyelitis optica, neuromyotonia, neutropenia,non-cancerous lymphocytosis, nongranulomatous uveitis, non-malignantthymoma, ocular and orbital inflammatory disorders, ocular cicatricialpemphigoid, oophoritis, ophthalmia symphatica, opsoclonus myoclonussyndrome (OMS), opsoclonus or opsoclonus myoclonus syndrome (OMS), andsensory neuropathy, optic neuritis, orchitis granulomatosa,osteoarthritis, palindromic rheumatism, pancreatitis, pancytopenia,PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated withStreptococcus), paraneoplastic cerebellar degeneration, paraneoplasticsyndrome, paraneoplastic syndromes, including neurologic paraneoplasticsyndromes, optionally Lambert-Eaton myasthenic syndrome or Eaton-Lambertsyndrome, parasitic diseases such as Lesihmania, paroxysmal nocturnalhemoglobinuria (PNH), Parry Romberg syndrome, pars planitis (peripheraluveitis), Parsonnage-Turner syndrome, parvovirus infection, pemphigoidsuch as pemphigoid bullous and skin pemphigoid, pemphigus (includingpemphigus vulgaris), pemphigus erythematosus, pemphigus foliaceus,pemphigus mucus-membrane pemphigoid, pemphigus, peptic ulcer, periodicparalysis, peripheral neuropathy, perivenous encephalomyelitis,pernicious anemia (anemia perniciosa), pernicious anemia, phacoantigenicuveitis, pneumonocirrhosis, POEMS syndrome, polyarteritis nodosa, TypeI, II, & III, polyarthritis chronica primaria, polychondritis (e.g.,refractory or relapsed polychondritis), polyendocrine autoimmunedisease, polyendocrine failure, polyglandular syndromes, optionallyautoimmune polyglandular syndromes (or polyglandular endocrinopathysyndromes), polymyalgia rheumatica, polymyositis,polymyositis/dermatomyositis, polyneuropathies, polyradiculitis acuta,post-cardiotomy syndrome, posterior uveitis, or autoimmune uveitis,postmyocardial infarction syndrome, postpericardiotomy syndrome,post-streptococcal nephritis, post-vaccination syndromes, preseniledementia, primary biliary cirrhosis, primary hypothyroidism, primaryidiopathic myxedema, primary lymphocytosis, which includes monoclonal Bcell lymphocytosis, optionally benign monoclonal gammopathy andmonoclonal gammopathy of undetermined significance, MGUS, primarymyxedema, primary progressive MS (PPMS), and relapsing remitting MS(RRMS), primary sclerosing cholangitis, progesterone dermatitis,progressive systemic sclerosis, proliferative arthritis, psoriasis suchas plaque psoriasis, psoriasis, psoriatic arthritis, pulmonary alveolarproteinosis, pulmonary infiltration eosinophilia, pure red cell anemiaor aplasia (PRCA), pure red cell aplasia, purulent or nonpurulentsinusitis, pustular psoriasis and psoriasis of the nails, pyelitis,pyoderma gangrenosum, Quervain's thyreoiditis, Raynauds phenomenon,reactive arthritis, recurrent abortion, reduction in blood pressureresponse, reflex sympathetic dystrophy, refractory sprue, Reiter'sdisease or syndrome, relapsing polychondritis, reperfusion injury ofmyocardial or other tissues, reperfusion injury, respiratory distresssyndrome, restless legs syndrome, retinal autoimmunity, retroperitonealfibrosis, Reynaud's syndrome, rheumatic diseases, rheumatic fever,rheumatism, rheumatoid arthritis, rheumatoid spondylitis, rubella virusinfection, Sampter's syndrome, sarcoidosis, schistosomiasis, Schmidtsyndrome, SCID and Epstein-Barr virus-associated diseases, sclera,scleritis, sclerodactyl, scleroderma, optionally systemic scleroderma,sclerosing cholangitis, sclerosis disseminata, sclerosis such assystemic sclerosis, sensoneural hearing loss, seronegativespondyloarthritides, Sheehan's syndrome, Shulman's syndrome, silicosis,Sjogren's syndrome, sperm & testicular autoimmunity, sphenoid sinusitis,Stevens-Johnson syndrome, stiff-man (or stiff-person) syndrome, subacutebacterial endocarditis (SBE), subacute cutaneous lupus erythematosus,sudden hearing loss, Susac's syndrome, Sydenham's chorea, sympatheticophthalmia, systemic lupus erythematosus (SLE) or systemic lupuserythematodes, cutaneous SLE, systemic necrotizing vasculitis,ANCA-associated vasculitis, optionally Churg-Strauss vasculitis orsyndrome (CSS), tabes dorsalis, Takayasu's arteritis, telangiectasia,temporal arteritis/Giant cell arteritis, thromboangitis ubiterans,thrombocytopenia, including thrombotic thrombocytopenic purpura (TTP)and autoimmune or immune-mediated thrombocytopenia such as idiopathicthrombocytopenic purpura (ITP) including chronic or acute ITP,thrombocytopenic purpura (TTP), thyrotoxicosis, tissue injury,Tolosa-Hunt syndrome, toxic epidermal necrolysis, toxic-shock syndrome,transfusion reaction, transient hypogammaglobulinemia of infancy,transverse myelitis, traverse myelitis, tropical pulmonary eosinophilia,tuberculosis, ulcerative colitis, undifferentiated connective tissuedisease (UCTD), urticaria, optionally chronic allergic urticaria andchronic idiopathic urticaria, including chronic autoimmune urticaria,uveitis, anterior uveitis, uveoretinitis, valvulitis, vasculardysfunction, vasculitis, vertebral arthritis, vesiculobullousdermatosis, vitiligo, Wegener's granulomatosis (Granulomatosis withPolyangiitis (GPA)), Wiskott-Aldrich syndrome, or x-linked hyper IgMsyndrome.

In a further embodiment, the method, use, or composition may furthercomprise the administration of another immune modulator and/or anantigen. In another embodiment, the immune modulator may be a TLRagonist (TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10,TLR11 agonist), may be a type 1 interferon, optionally alpha interferonor beta interferon, or a CD40 agonist.

In one embodiment, the method of treating an inflammatory disorder maycomprise administering an effective amount of a VISTA fusion protein,optionally a VISTA-Ig, or a multimeric VISTA protein.

In one embodiment, the composition for treating an inflammatory disordermay comprise an effective amount of a VISTA fusion protein, optionally aVISTA-Ig, or a multimeric VISTA protein.

In one embodiment, the use of an effective amount of a VISTA fusionprotein, optionally a VISTA-Ig, or a multimeric VISTA protein for themanufacture of a medicament for the treatment of an inflammatorydisorder.

In one embodiment, the disorder treated may be selected from type 1diabetes, multiple sclerosis, rheumatoid arthritis, psoriatic arthritis,systemic lupus erythematosis, rheumatic diseases, allergic disorders,asthma, allergic rhinitis, skin disorders, Crohn's disease, ulcerativecolitis, transplant rejection, graft-versus-host disease,poststreptococcal and autoimmune renal failure, septic shock, systemicinflammatory response syndrome (SIRS), adult respiratory distresssyndrome (ARDS) and envenomation; autoinflammatory diseases,osteoarthritis, crystal arthritis, capsulitis, arthropathies,tendonitis, ligamentitis or traumatic joint injury.

In one embodiment, the disorder treated may be multiple sclerosis orrheumatoid arthritis.

In one embodiment, the method of treating graft-versus-host-disease(GVHD) may comprise administration of an effective amount of aneffective amount of a VISTA fusion protein, optionally a VISTA-Ig, or amultimeric VISTA protein.

In one embodiment, the composition for treatinggraft-versus-host-disease (GVHD) may comprise administration of aneffective amount of an effective amount of a VISTA fusion protein,optionally a VISTA-Ig, or a multimeric VISTA protein.

In one embodiment, the use of an effective amount of an effective amountof a VISTA fusion protein, optionally a VISTA-Ig, or a multimeric VISTAprotein in the manufacture of a medicament for the treatment ofgraft-versus-host-disease (GVHD).

In one embodiment, the graft-versus-host-disease may be acutegraft-versus-host disease, chronic graft-versus-host disease, acutegraft-versus-host disease associated with stem cell transplant, chronicgraft-versus-host disease associated with stem cell transplant, acutegraft-versus-host disease associated with bone marrow transplant, acutegraft-versus-host disease associated with allogeneic hemapoetic stemcell transplant (HSCT), or chronic graft-versus-host disease associatedwith bone marrow transplant.

In one embodiment, the patient treated may have at least one symptom ofgraft-versus-host disease (GVHD), optionally wherein the patientexhibits acute GVHD includes but is not limited to abdominal pain,abdominal cramps, diarrhea, fever, jaundice, skin rash, vomiting, andweight loss. In one embodiment, the patient treated may have at leastone symptom of chronic graft-versus-host disease (GVHD) includes but isnot limited to dry eyes, dry mouth, hair loss, hepatisis, lung disorder,gastrointestinal tract disorders, skin rash, and skin thickening. In oneembodiment, the patient has or is to receive allogeneic stem cell orbone marrow transplant.

In one embodiment, the patient may have or is to receive autologous stemcell or bone marrow transplant.

In one embodiment, the method of treating an individual with anallergic, inflammatory or autoimmune disorder may comprise administeringan effective amount of a VISTA fusion protein, optionally a VISTA-Ig, ora multimeric VISTA protein.

In one embodiment, the composition for treating an individual with anallergic, inflammatory or autoimmune disorder may comprise administeringan effective amount of a VISTA fusion protein, optionally a VISTA-Ig, ora multimeric VISTA protein.

In one embodiment, the use of an effective amount of a VISTA fusionprotein, optionally a VISTA-Ig, or a multimeric VISTA protein for themanufacture of a medicament for the treatment of an allergic,inflammatory or autoimmune disorder.

In another embodiment, the allergic, inflammatory or autoimmune disordermay be selected from psoriasis, dermatitis, atopic dermatitis; systemicscleroderma, sclerosis; Crohn's disease, ulcerative colitis; respiratorydistress syndrome, adult respiratory distress syndrome; ARDS);dermatitis; meningitis; encephalitis; uveitis; colitis;glomerulonephritis; eczema, asthma, atherosclerosis; leukocyte adhesiondeficiency; rheumatoid arthritis; systemic lupus erythematosus (SLE);diabetes mellitus, optionally Type I diabetes mellitus or insulindependent diabetes mellitis; multiple sclerosis; Reynaud's syndrome;autoimmune thyroiditis; allergic encephalomyelitis; Sjorgen's syndrome;juvenile onset diabetes; tuberculosis, sarcoidosis, polymyositis,granulomatosis and vasculitis; pernicious anemia (Addison's disease);graft rejection disease, GVHD, central nervous system (CNS) inflammatorydisorder; multiple organ injury syndrome; hemolytic anemia,cryoglobinemia or Coombs positive anemia; myasthenia gravis;antigen-antibody complex mediated diseases; anti-glomerular basementmembrane disease; antiphospholipid syndrome; allergic neuritis; Graves'disease; Lambert-Eaton myasthenic syndrome; pemphigoid bullous;pemphigus; autoimmune polyendocrinopathies; Reiter's disease; stiff-mansyndrome; Behcet disease; giant cell arteritis; immune complexnephritis; IgA nephropathy; IgM polyneuropathies; immunethrombocytopenic purpura (ITP) and autoimmune thrombocytopenia.

In another embodiment, the disease may be selected from arthritis,rheumatoid arthritis, acute arthritis, chronic rheumatoid arthritis,gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis,degenerative arthritis, infectious arthritis, Lyme arthritis,proliferative arthritis, psoriatic arthritis, vertebral arthritis, andjuvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronicaprogrediente, arthritis deformans, polyarthritis chronica primaria,reactive arthritis, and ankylosing spondylitis), inflammatoryhyperproliferative skin diseases, psoriasis such as plaque psoriasis,gutatte psoriasis, pustular psoriasis, and psoriasis of the nails,dermatitis including contact dermatitis, chronic contact dermatitis,allergic dermatitis, allergic contact dermatitis, dermatitisherpetiformis, and atopic dermatitis, x-linked hyper IgM syndrome,urticaria such as chronic allergic urticaria and chronic idiopathicurticaria, including chronic autoimmune urticaria,polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermalnecrolysis, scleroderma, systemic scleroderma, sclerosis, systemicsclerosis, multiple sclerosis (MS), spino-optical MS, primaryprogressive MS (PPMS), relapsing remitting MS (RRMS), progressivesystemic sclerosis, atherosclerosis, arteriosclerosis, sclerosisdisseminata, and ataxic sclerosis, inflammatory bowel disease (IBD),Crohn's disease, colitis, ulcerative colitis, colitis ulcerosa,microscopic colitis, collagenous colitis, colitis polyposa, necrotizingenterocolitis, transmural colitis, autoimmune inflammatory boweldisease, pyoderma gangrenosum, erythema nodosum, primary sclerosingcholangitis, episcleritis, respiratory distress syndrome, adult or acuterespiratory distress syndrome (ARDS), meningitis, inflammation of all orpart of the uvea, iritis, choroiditis, an autoimmune hematologicaldisorder, rheumatoid spondylitis, sudden hearing loss, IgE-mediateddiseases such as anaphylaxis and allergic and atopic rhinitis,encephalitis, Rasmussen's encephalitis, limbic and/or brainstemencephalitis, uveitis, anterior uveitis, acute anterior uveitis,granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis,posterior uveitis, autoimmune uveitis, glomerulonephritis (GN),idiopathic membranous GN or idiopathic membranous nephropathy, membrano-or membranous proliferative GN (MPGN), rapidly progressive GN, allergicconditions, autoimmune myocarditis, leukocyte adhesion deficiency,systemic lupus erythematosus (SLE) or systemic lupus erythematodes suchas cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal lupussyndrome (NLE), lupus erythematosus disseminatus, lupus (includingnephritis, cerebritis, pediatric, non-renal, extra-renal, discoid,alopecia), juvenile onset (Type I) diabetes mellitus, includingpediatric insulin-dependent diabetes mellitus (IDDM), adult onsetdiabetes mellitus (Type II diabetes), autoimmune diabetes, idiopathicdiabetes insipidus, immune responses associated with acute and delayedhypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis,sarcoidosis, granulomatosis, lymphomatoid granulomatosis, Wegener'sgranulomatosis, agranulocytosis, vasculitides, including vasculitis,large vessel vasculitis, polymyalgia rheumatica, giant cell (Takayasu's)arteritis, medium vessel vasculitis, Kawasaki's disease, polyarteritisnodosa, microscopic polyarteritis, CNS vasculitis, necrotizing,cutaneous, hypersensitivity vasculitis, systemic necrotizing vasculitis,and ANCA-associated vasculitis, such as Churg-Strauss vasculitis orsyndrome (CSS), temporal arteritis, aplastic anemia, autoimmune aplasticanemia, Coombs positive anemia, Diamond Blackfan anemia, hemolyticanemia or immune hemolytic anemia including autoimmune hemolytic anemia(AIHA), pernicious anemia (anemia perniciosa), Addison's disease, purered cell anemia or aplasia (PRCA), Factor VIII deficiency, hemophilia A,autoimmune neutropenia, pancytopenia, leukopenia, diseases involvingleukocyte diapedesis, CNS inflammatory disorders, multiple organ injurysyndrome such as those secondary to septicemia, trauma or hemorrhage,antigen-antibody complex-mediated diseases, anti-glomerular basementmembrane disease, anti-phospholipid antibody syndrome, allergicneuritis, Bechet's or Behcet's disease, Castleman's syndrome,Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome,Stevens-Johnson syndrome, pemphigoid such as pemphigoid bullous and skinpemphigoid, pemphigus, optionally pemphigus vulgaris, pemphigusfoliaceus, pemphigus mucus-membrane pemphigoid, pemphigus erythematosus,autoimmune polyendocrinopathies, Reiter's disease or syndrome, immunecomplex nephritis, antibody-mediated nephritis, neuromyelitis optica,polyneuropathies, chronic neuropathy, IgM polyneuropathies, IgM-mediatedneuropathy, thrombocytopenia, thrombotic thrombocytopenic purpura (TTP),idiopathic thrombocytopenic purpura (ITP), autoimmune orchitis andoophoritis, primary hypothyroidism, hypoparathyroidism, autoimmunethyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto'sthyroiditis); subacute thyroiditis, autoimmune thyroid disease,idiopathic hypothyroidism, Grave's disease, polyglandular syndromes suchas autoimmune polyglandular syndromes (or polyglandular endocrinopathysyndromes), paraneoplastic syndromes, including neurologicparaneoplastic syndromes such as Lambert-Eaton myasthenic syndrome orEaton-Lambert syndrome, stiff-man or stiff-person syndrome,encephalomyelitis, allergic encephalomyelitis, experimental allergicencephalomyelitis (EAE), myasthenia gravis, thymoma-associatedmyasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus oropsoclonus myoclonus syndrome (OMS), and sensory neuropathy, multifocalmotor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronichepatitis, lupoid hepatitis, giant cell hepatitis, chronic activehepatitis or autoimmune chronic active hepatitis, lymphoid interstitialpneumonitis, bronchiolitis obliterans (non-transplant) vs NSIP,Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathicIgA nephropathy, linear IgA dermatosis, primary biliary cirrhosis,pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac disease,Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue,idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS;Lou Gehrig's disease), coronary artery disease, autoimmune ear diseasesuch as autoimmune inner ear disease (AGED), autoimmune hearing loss,opsoclonus myoclonus syndrome (OMS), polychondritis such as refractoryor relapsed polychondritis, pulmonary alveolar proteinosis, amyloidosis,scleritis, a non-cancerous lymphocytosis, a primary lymphocytosis, whichincludes monoclonal B cell lymphocytosis, optionally benign monoclonalgammopathy or monoclonal garnmopathy of undetermined significance, MGUS,peripheral neuropathy, paraneoplastic syndrome, channelopathies such asepilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness,periodic paralysis, and channelopathies of the CNS, autism, inflammatorymyopathy, focal segmental glomerulosclerosis (FSGS), endocrineopthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatologicaldisorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome,adrenalitis, gastric atrophy, presenile dementia, demyelinating diseasessuch as autoimmune demyelinating diseases, diabetic nephropathy,Dressler's syndrome, alopecia greata, CREST syndrome (calcinosis,Raynaud's phenomenon, esophageal dysmotility, sclerodactyl), andtelangiectasia), male and female autoimmune infertility, mixedconnective tissue disease, Chagas' disease, rheumatic fever, recurrentabortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome,Cushing's syndrome, bird-fancier's lung, allergic granulomatousangiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitissuch as allergic alveolitis and fibrosing alveolitis, interstitial lungdisease, transfusion reaction, leprosy, malaria, leishmaniasis,kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter'ssyndrome, Caplan's syndrome, dengue, endocarditis, endomyocardialfibrosis, diffuse interstitial pulmonary fibrosis, interstitial lungfibrosis, idiopathic pulmonary fibrosis, cystic fibrosis,endophthalmitis, erythema elevatum et diutinum, erythroblastosisfetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome,flariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis,iridocyclitis, or Fuch's cyclitis, Henoch-Schonlein purpura, humanimmunodeficiency virus (HIV) infection, echovirus infection,cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virusinfection, post-vaccination syndromes, congenital rubella infection,Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadalfailure, Sydenham's chorea, post-streptococcal nephritis, thromboangitisubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant cellpolymyalgia, endocrine ophthamopathy, chronic hypersensitivitypneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis,idiopathic nephritic syndrome, minimal change nephropathy, benignfamilial and ischemia-reperfusion injury, retinal autoimmunity, jointinflammation, bronchitis, chronic obstructive airway disease, silicosis,aphthae, aphthous stomatitis, arteriosclerotic disorders,aspermiogenese, autoimmune hemolysis, Boeck's disease, cryoglobulinemia,Dupuytren's contracture, endophthalmia phacoanaphylactica, enteritisallergica, erythema nodosum leprosum, idiopathic facial paralysis,chronic fatigue syndrome, febris rheumatica, Hamman-Rich's disease,sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism,ileitis regionalis, leucopenia, mononucleosis infectiosa, traversemyelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica,orchitis granulomatosa, pancreatitis, polyradiculitis acuta, pyodermagangrenosum, Quervain's thyreoiditis, acquired spenic atrophy,infertility due to antispermatozoan antobodies, non-malignant thymoma,vitiligo, SCID and Epstein-Barr virus-associated diseases, acquiredimmune deficiency syndrome (AIDS), parasitic diseases such asLesihmania, toxic-shock syndrome, food poisoning, conditions involvinginfiltration of T cells, leukocyte-adhesion deficiency, immune responsesassociated with acute and delayed hypersensitivity mediated by cytokinesand T-lymphocytes, diseases involving leukocyte diapedesis, multipleorgan injury syndrome, antigen-antibody complex-mediated diseases,antiglomerular basement membrane disease, allergic neuritis, autoimmunepolyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophicgastritis, sympathetic ophthalmia, rheumatic diseases, mixed connectivetissue disease, nephrotic syndrome, insulitis, polyendocrine failure,peripheral neuropathy, autoimmune polyglandular syndrome type I,adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis,dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA),hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosingcholangitis, purulent or nonpurulent sinusitis, acute or chronicsinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, aneosinophil-related disorder such as eosinophilia, pulmonary infiltrationeosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chroniceosinophilic pneumonia, tropical pulmonary eosinophilia,bronchopneumonic aspergillosis, aspergilloma, or granulomas containingeosinophils, anaphylaxis, seronegative spondyloarthritides,polyendocrine autoimmune disease, sclerosing cholangitis, sclera,episclera, chronic mucocutaneous candidiasis, Bruton's syndrome,transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome,ataxia telangiectasia, autoimmune disorders associated with collagendisease, rheumatism, neurological disease, ischemic re-perfusiondisorder, reduction in blood pressure response, vascular dysfunction,antgiectasis, tissue injury, cardiovascular ischemia, hyperalgesia,cerebral ischemia, and disease accompanying vascularization, allergichypersensitivity disorders, glomerulonephritides, reperfusion injury,reperfusion injury of myocardial or other tissues, dermatoses with acuteinflammatory components, acute purulent meningitis or other centralnervous system inflammatory disorders, ocular and orbital inflammatorydisorders, granulocyte transfusion-associated syndromes,cytokine-induced toxicity, acute serious inflammation, chronicintractable inflammation, pyelitis, pneumonocirrhosis, diabeticretinopathy, diabetic large-artery disorder, endarterial hyperplasia,peptic ulcer, valvulitis, and endometriosis.

In one embodiment, a method of making antibodies may comprise immunizingan animal with a VISTA epitope, removing said animal's spleen andprepare a single cell suspension, fusing a spleen cell with a myelomacell, culturing post-fusion cells in hybridoma selection medium,culturing the resultant hybridomas, screening for specific antibodyproduction, and selecting hybridomas which produce the desired antibody.

In another embodiment, an anti-VISTA or antibody fragment thereofproduced by the method comprising immunizing an animal with a VISTAepitope, removing said animal's spleen and prepare a single cellsuspension, fusing a spleen cell with a myeloma cell, culturingpost-fusion cells in hybridoma selection medium, culturing the resultanthybridomas, screening for specific antibody production, and selectinghybridomas which produce the desired antibody.

In another embodiment, the anti-VISTA or antibody fragment thereof maybe a humanized, chimeric, or single chain variant.

In one embodiment, an isolated VISTA antagonist may be an antibody or anantibody fragment thereof, a peptide, a glycoalkoid, an antisensenucleic acid, a ribozyme, a retinoid, an avemir, a small molecule, orany combination thereof.

In one embodiment, a method of treating or preventing inflammation in asubject in need thereof may comprise administering an effective amountof an isolated VISTA antagonist, wherein said antagonist may be anantibody or an antibody fragment thereof, a peptide, a glycoalkoid, anantisense nucleic acid, a ribozyme, a retinoid, an avemir, a smallmolecule, or any combination thereof.

In one embodiment, a method of treating an autoimmune disease maycomprise administering an effective amount of an isolated VISTAantagonist, wherein said antagonist may be an antibody or an antibodyfragment thereof, a peptide, a glycoalkoid, an antisense nucleic acid, aribozyme, a retinoid, an avemir, a small molecule, or any combinationthereof.

In one embodiment, a method of treating an inflammatory disorder maycomprise administering an effective amount of an isolated VISTAantagonist, wherein said antagonist may be an antibody or an antibodyfragment thereof, a peptide, a glycoalkoid, an antisense nucleic acid, aribozyme, a retinoid, an avemir, a small molecule, or any combinationthereof.

In one embodiment, a method of treating graft-versus-host-disease (GVHD)may comprise administration of an effective amount of an effectiveamount of an isolated VISTA antagonist, wherein said antagonist may bean antibody or an antibody fragment thereof, a peptide, a glycoalkoid,an antisense nucleic acid, a ribozyme, a retinoid, an avemir, a smallmolecule, or any combination thereof.

In one embodiment, a method of treating an individual with an allergic,inflammatory or autoimmune disorder may comprise administering aneffective amount of an isolated VISTA antagonist, wherein saidantagonist may be an antibody or an antibody fragment thereof, apeptide, a glycoalkoid, an antisense nucleic acid, a ribozyme, aretinoid, an avemir, a small molecule, or any combination thereof.

In one embodiment, a composition for treating or preventing inflammationin a subject in need thereof may comprise administering an effectiveamount of an isolated VISTA antagonist, wherein said antagonist may bean antibody or an antibody fragment thereof, a peptide, a glycoalkoid,an antisense nucleic acid, a ribozyme, a retinoid, an avemir, a smallmolecule, or any combination thereof.

In one embodiment, a composition for treating an autoimmune disease maycomprise administering an effective amount of an isolated VISTAantagonist, wherein said antagonist may be an antibody or an antibodyfragment thereof, a peptide, a glycoalkoid, an antisense nucleic acid, aribozyme, a retinoid, an avemir, a small molecule, or any combinationthereof.

In one embodiment, a composition for treating an inflammatory disordermay comprise administering an effective amount of an isolated VISTAantagonist, wherein said antagonist may be an antibody or an antibodyfragment thereof, a peptide, a glycoalkoid, an antisense nucleic acid, aribozyme, a retinoid, an avemir, a small molecule, or any combinationthereof.

In one embodiment, a composition for treating graft-versus-host-disease(GVHD) may comprise administration of an effective amount of aneffective amount of an isolated VISTA antagonist, wherein saidantagonist may be an antibody or an antibody fragment thereof, apeptide, a glycoalkoid, an antisense nucleic acid, a ribozyme, aretinoid, an avemir, a small molecule, or any combination thereof.

In one embodiment, a composition for treating an individual with anallergic, inflammatory or autoimmune disorder may comprise administeringan effective amount of an isolated VISTA antagonist, wherein saidantagonist may be an antibody or an antibody fragment thereof, apeptide, a glycoalkoid, an antisense nucleic acid, a ribozyme, aretinoid, an avemir, a small molecule, or any combination thereof.

In one embodiment, the use of an effective amount of an isolated VISTAantagonist, wherein said antagonist may be an antibody or an antibodyfragment thereof, a peptide, a glycoalkoid, an antisense nucleic acid, aribozyme, a retinoid, an avemir, a small molecule, or any combinationthereof, for the manufacture of a medicament for treating or preventinginflammation.

In one embodiment, the use of an effective amount of an effective amountof an isolated VISTA antagonist, wherein said antagonist may be anantibody or an antibody fragment thereof, a peptide, a glycoalkoid, anantisense nucleic acid, a ribozyme, a retinoid, an avemir, a smallmolecule, or any combination thereof, for the manufacture of amedicament for the treatment of an autoimmune disease.

In one embodiment, the use of an effective amount of an effective amountof an isolated VISTA antagonist, wherein said antagonist may be anantibody or an antibody fragment thereof, a peptide, a glycoalkoid, anantisense nucleic acid, a ribozyme, a retinoid, an avemir, a smallmolecule, or any combination thereof, for the manufacture of amedicament for the treatment of an inflammatory disorder.

In one embodiment, the use of an effective amount of an effective amountof an isolated VISTA antagonist, wherein said antagonist may be anantibody or an antibody fragment thereof, a peptide, a glycoalkoid, anantisense nucleic acid, a ribozyme, a retinoid, an avemir, a smallmolecule, or any combination thereof, for the manufacture of amedicament for the treatment of graft-versus-host-disease (GVHD).

In one embodiment, the use of an effective amount of an effective amountof an isolated VISTA antagonist, wherein said antagonist may be anantibody or an antibody fragment thereof, a peptide, a glycoalkoid, anantisense nucleic acid, a ribozyme, a retinoid, an avemir, a smallmolecule, or any combination thereof, for the manufacture of amedicament for the treatment of an allergic, inflammatory or autoimmunedisorder.

In one embodiment, a method for detecting VISTA in a sample may comprisecontacting a sample with an anti-VISTA antibody or antibody fragment anddetecting the anti-VISTA antibody-VISTA conjugates. In anotherembodiment, the sample may be a biological sample. In anotherembodiment, the anti-VISTA antibody binds the amino acid sequence of SEQID NO: 2, 3, or 5.

In another embodiment, compositions for therapeutic, diagnostic orimmune modulatory usage may comprise an isolated soluble VISTA (PD-L3)protein or VISTA fusion protein (e.g., a soluble VISTA-Ig fusion proteinor a multimeric VISTA protein) may comprise an amino acid sequence thatpreferably may be at least 70-90% identical to the human or murine VISTA(PD-L3) polypeptide set forth in SEQ ID NO: 2, 4 or 5 or an ortholog, orfragment thereof encoded by a gene that specifically hybridizes to SEQID NO:1 or 3 that modulates VISTA in vivo and a pharmaceuticallyacceptable carrier. In some embodiments, the soluble or multimeric VISTAprotein may be directly or indirectly linked to a heterologous(non-VISTA) protein or may be expressed by a viral vector or a cellcontaining (e.g., a transfected immune cell such as a T cell.)

In an embodiment, isolated or recombinant VISTA (PD-L3) polypeptides(e.g., proteins, polypeptides, peptides, or fragments or portionsthereof). In one embodiment, an isolated VISTA (PD-L3) polypeptide orVISTA (PD-L3) fusion protein comprises at least one of the followingdomains: a signal peptide domain, an IgV domain, an extracellulardomain, a transmembrane domain, or a cytoplasmic domain.

In an embodiment, a VISTA (PD-L3) polypeptide comprises at least one ofthe following domains: a signal peptide domain, an IgV domain, anextracellular domain, a transmembrane domain, or a cytoplasmic domain,and comprises an amino acid sequence at least about 71%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the aminoacid sequence of SEQ ID NO: 2, 4, or 5. In another embodiment, a VISTA(PD-L3) polypeptide comprises at least one of the following domains: asignal peptide domain, an IgV domain, an extracellular domain, atransmembrane domain, or a cytoplasmic domain, and may have a VISTA(PD-L3) activity (as described herein).

In one embodiment, an isolated VISTA protein may comprise a polypeptidewith at least about 90% sequence identity to the extracellular domain ofthe polypeptide sequence of SEQ ID NO: 2, 4, 5, 16-25, 36, or 37. In afurther embodiment, the polypeptide may have at least about 95% sequenceidentity to the polypeptide sequence of SEQ ID NO: 2, 4, 5, 16-25, 36,or 37.

In another embodiment, a VISTA polypeptide comprises at least one of thefollowing domains: a signal peptide domain, an IgV domain, anextracellular domain, a transmembrane domain, or a cytoplasmic domain,and may be encoded by a nucleic acid molecule having a nucleotidesequence which hybridizes under stringent hybridization conditions to acomplement of a nucleic acid molecule may comprise the nucleotidesequence of SEQ ID NO: 1 or 3.

In another embodiment, fragments or portions of the polypeptide maycomprise the amino acid sequence of SEQ ID NO: 2, 4, or 5, wherein thefragment comprises at least 15 amino acids (i.e., contiguous aminoacids) of the amino acid sequence of SEQ ID NO: 2 or 4. In anotherembodiment, a VISTA (PD-L3) polypeptide comprises or consists of theamino acid sequence of SEQ ID NO: 2, 4 or 5. In another embodiment, aVISTA (PD-L3) polypeptide may be encoded by a nucleic acid molecule maycomprise a nucleotide sequence at least about 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to a nucleotidesequence of SEQ ID NO: 1 or 3, or a complement thereof. A VISTA (PD-L3)polypeptide which may be encoded by a nucleic acid molecule consistingof a nucleotide sequence which hybridizes under stringent hybridizationconditions to a complement of a nucleic acid molecule may comprise thenucleotide sequence of SEQ ID NO: 1 or 3.

In one embodiment, the VISTA polypeptides may be agonists wherein theyinduce suppression. In another embodiment, the VISTA polypeptides may beantagonists wherein they interfere with suppression.

The polypeptides of the present invention or portions thereof, e.g.,biologically active portions thereof, may be operatively linked to anon-VISTA (PD-L3) polypeptide (e.g., heterologous amino acid sequences)to form fusion polypeptides.

In one embodiment, expression vectors may comprise an isolated nucleicacid encoding a VISTA protein that may be at least about 70-99%identical to the human or murine VISTA amino acid sequence set forth inSEQ ID NO: 2, 4 or 5 or a fragment or ortholog thereof, which optionallymay be fused to a sequence encoding another protein such as an Igpolypeptide (e.g., an Fc region) or a reporter molecule; and host cellscontaining said vectors.

In another embodiment, isolated nucleic acid molecules encoding VISTApolypeptides, preferably encoding soluble fusion proteins and multimericVISTA proteins as well as nucleic acid fragments suitable as primers orhybridization probes for the detection of VISTA (PD-L3)-encoding nucleicacids. In one embodiment, a VISTA (PD-L3) nucleic acid molecule of theinvention may be at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence (e.g.,to the entire length of the nucleotide sequence) encoding VISTA (PD-L3)in SEQ ID NO:1 or 3 or a complement thereof.

In another embodiment, a VISTA (PD-L3) nucleic acid molecule comprises anucleotide sequence encoding a polypeptide having an amino acid sequencehaving a specific percent identity to the amino acid sequence of SEQ IDNO: 2, 4 or 5. In an embodiment, a VISTA (PD-L3) nucleic acid moleculecomprises a nucleotide sequence encoding a polypeptide having an aminoacid sequence at least about 71%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% identical to the entire length of the amino acid sequence ofSEQ ID NO: 2, 4 or 5 or to the extracellular domain thereof.

In another embodiment, an isolated nucleic acid molecule encodes theamino acid sequence of human or murine or VISTA or a conserved region orfunctional domain therein. In yet another embodiment, the nucleic acidmolecule comprises a nucleotide sequence encoding a polypeptide maycomprise the amino acid sequence of SEQ ID NO: 2, 4 or 5. In yet anotherembodiment, the nucleic acid molecule may be at least about 50, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000, 1050, 1100, 1150 nucleotides in length. In afurther embodiment, the nucleic acid molecule may be at least about 50,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, 1000, 1050, 1100, 1150 nucleotides in length andencodes a polypeptide having a VISTA (PD-L3) activity or modulatingVISTA (PD-L3) function.

Another embodiment features nucleic acid molecules, preferably VISTA(PD-L3) nucleic acid molecules, which specifically detect VISTA (PD-L3)nucleic acid molecules relative to nucleic acid molecules encodingnon-VISTA (PD-L3) polypeptides. For example, in one embodiment, anucleic acid molecule may be at least about 880, 900, 950, 1000, 1050,1100, 1150 nucleotides in length and hybridizes under stringentconditions to a nucleic acid molecule encoding the polypeptide shown inSEQ ID NO: 2, 4 or 5, or a complement thereof. In another embodiment, anucleic acid molecule may be at least 20, 30, 40, 50, 100, 150, 200,250, 300 nucleotides in length and hybridizes under stringent conditionsto a nucleic acid molecule encoding a fragment of VISTA (PD-L3), e.g.,may comprise at least about 20, 30, 40, 50, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950nucleotides in length, comprises at least 15 (i.e., 15 contiguous)nucleotides of the disclosed nucleic acid sequence in SEQ ID NO:1 and 3encoding the VISTA (PD-L3) polypeptides in SEQ ID NO: 2, 4 or 5, or acomplement thereof, and hybridizes under stringent conditions to anucleic acid molecule may comprise the nucleotide sequence shown in SEQID NO: 1 or 3 or a complement thereof.

In one embodiment, the nucleic acid molecule encodes a naturallyoccurring allelic variant of a polypeptide may comprise the amino acidsequence of SEQ ID NO: 2 or 4 or 5, wherein the nucleic acid moleculehybridizes to a complement of a nucleic acid molecule may comprise SEQID NO: 1 or 3, or a complement thereof, under stringent conditions.

Another embodiment of the invention provides an isolated antisense to aVISTA (PD-L3) nucleic acid molecule (e.g., antisense to the codingstrand of a VISTA (PD-L3) nucleic acid molecule of SEQ ID NO: 1 or 3.)

Another aspect of the invention provides a vector may comprise a VISTA(PD-L3) nucleic acid molecule. In certain embodiments, the vector may bea recombinant expression vector.

In another embodiment, a host cell comprises a vector of the invention.In yet another embodiment, a host cell comprises a nucleic acid moleculeof the invention. The invention also provides a method for producing apolypeptide, preferably a VISTA (PD-L3) polypeptide, by culturing in asuitable medium, a host cell, e.g., a mammalian host cell such as anon-human mammalian cell, of the invention containing a recombinantexpression vector, such that the polypeptide may be produced.

In one embodiment, an siRNA molecule which targets VISTA mRNAtranscribed from a VISTA DNA may comprise the nucleic acid sequence ofSEQ ID NO: 1 or 3. In another embodiment, an siRNA molecule whichtargets VISTA mRNA transcribed from a VISTA DNA encoding the amino acidsequence set forth in SEQ ID NO: 2, 4 or 5. In a further embodiment, ansiRNA molecule that targets VISTA may comprise the nucleic acid sequenceof any one of SEQ ID NOs: 38-67. In another embodiment, an siRNAmolecule that targets either the ORF or UTR region of VISTA may comprisethe amino acid sequence of any one of SEQ ID NO: 38-47. In anotherembodiment, an siRNA molecule that targets the UTR region only of VISTAmay comprise the amino acid sequence of any one of SEQ ID NO: 48-57. Inanother embodiment, an siRNA molecule that targets the ORF region onlyof VISTA may comprise the amino acid sequence of any one of SEQ ID NO:58-67. In one embodiment, an siRNA molecule that targets VISTA mayconsist of the nucleic acid sequence of any one of SEQ ID NOs: 38-67. Inone embodiment, an siRNA molecule that targets either the ORF or UTRregion of VISTA may consist of the amino acid sequence of any one of SEQID NO: 38-47. In one embodiment, an siRNA molecule that targets the UTRregion only of VISTA may consist the amino acid sequence of any one ofSEQ ID NO: 48-57. In one embodiment, an siRNA molecule that targets theORF region only of VISTA may consist the amino acid sequence of any oneof SEQ ID NO: 58-67.

In a further embodiment, a composition may comprise an siRNA moleculecomprising the nucleic acid sequence of any one of SEQ ID NOs: 38-67. Ina further embodiment, a composition may comprise an siRNA moleculeconsisting of the nucleic acid sequence of any one of SEQ ID NOs: 38-67.In a further embodiment, a composition may be a pharmaceuticalcomposition.

In one embodiment, a method for treating an autoimmune disorder maycomprise administration of an siRNA molecule that targets VISTAcomprising the nucleic acid sequence of any one of SEQ ID NOs: 38-67. Inone embodiment, a method for treating an autoimmune disorder maycomprise administration of an siRNA molecule that targets VISTAconsisting of the nucleic acid sequence of any one of SEQ ID NOs: 38-67.In a further embodiment, a composition for treating an autoimmunedisorder may comprise an siRNA molecule comprising the nucleic acidsequence of any one of SEQ ID NOs: 38-67. In a further embodiment, acomposition for treating an autoimmune disorder may comprise an siRNAmolecule consisting of the nucleic acid sequence of any one of SEQ IDNOs: 38-67. In a further embodiment, use of an siRNA molecule comprisingany one of nucleic acid sequences of SEQ ID NOs: 38-67 for themanufacture of a medicament for the treatment of an autoimmune disease.In a further embodiment, use of an siRNA molecule consisting of any oneof nucleic acid sequences of SEQ ID NOs: 38-67 for the manufacture of amedicament for the treatment of an autoimmune disease.

In one embodiment, a method for treating an inflammatory disorder maycomprise administration of an siRNA molecule that targets VISTAcomprising the nucleic acid sequence of any one of SEQ ID NOs: 38-67. Inone embodiment, a method for treating an inflammatory disorder maycomprise administration of an siRNA molecule that targets VISTAconsisting of the nucleic acid sequence of any one of SEQ ID NOs: 38-67.In a further embodiment, a composition for treating an inflammatorydisorder may comprise an siRNA molecule comprising the nucleic acidsequence of any one of SEQ ID NOs: 38-67. In a further embodiment, acomposition for treating an inflammatory disorder an siRNA molecule maycomprise an siRNA molecule consisting of the nucleic acid sequence ofany one of SEQ ID NOs: 38-67. In a further embodiment, use of an siRNAmolecule comprising any one of nucleic acid sequences of SEQ ID NOs:38-67 for the manufacture of a medicament for the treatment of aninflammatory disease. In a further embodiment, use of an siRNA moleculeconsisting of any one of nucleic acid sequences of SEQ ID NOs: 38-67 forthe manufacture of a medicament for the treatment of an inflammatorydisease.

In one embodiment, a method for treating graft-versus-host disease maycomprise administration of an siRNA molecule that targets VISTAcomprising the nucleic acid sequence of any one of SEQ ID NOs: 38-67. Inone embodiment, a method for treating graft-versus-host disease maycomprise administration of an siRNA molecule that targets VISTAconsisting of the nucleic acid sequence of any one of SEQ ID NOs: 38-67.In a further embodiment, a composition for treating graft-versus-hostdisease may comprise an siRNA molecule comprising the nucleic acidsequence of any one of SEQ ID NOs: 38-67. In a further embodiment, acomposition for treating graft-versus-host disease an siRNA molecule maycomprise an siRNA molecule consisting of the nucleic acid sequence ofany one of SEQ ID NOs: 38-67. In a further embodiment, use of an siRNAmolecule comprising any one of nucleic acid sequences of SEQ ID NOs:38-67 for the manufacture of a medicament for the treatment ofgraft-versus-host disease. In a further embodiment, use of an siRNAmolecule consisting of any one of nucleic acid sequences of SEQ ID NOs:38-67 for the manufacture of a medicament for the treatment ofgraft-versus-host disease.

In one embodiment, an antagonist may specifically binds to a VISTA(PD-L3) protein may comprise the amino acid sequence set forth in SEQ IDNO:2, 4 or 5 or a variant, fragment, or ortholog thereof. In anembodiment, the binding agent modulates (agonizes or antagonizes) VISTAactivity in vitro or in vivo.

In one embodiment, the VISTA antagonist may be a VISTA ligand. Inanother embodiment, the VISTA ligand may be a protein. In anotherembodiment, the VISTA antagonist may be an antibody or an antibodyfragment ther7eof, a peptide, a glycoalkoid, an antisense nucleic acid,a ribozyme, a retinoid, an avemir, a small molecule, or any combinationthereof.

In one embodiment, the VISTA antagonists may have functional propertiesincluding but not limited to modulating specific effects of VISTA(PD-L3) on immunity such as the suppressive effect of the protein on TCRactivation, the suppressive effect of the protein on CD4 T cellproliferative responses to anti-CD3, suppression of antigen specificproliferative responses of cognate CD4 T cells, the suppressive effectsof VISTA (PD-L3) on the expression of specific cytokines (e.g., IL-2 andγ interferon).

In one embodiment, an antagonist, optionally a proteinanceousantagonist, that specifically binds to a VISTA polypeptide, multimericVISTA polypeptide, or VISTA fusion protein. In another embodiment, theantagonist, optionally a proteinanceous antagonist, may exhibitantitumor or antimetastatic activity. In another embodiment, theantagonist, optionally a proteinanceous antagonist, may specificallybind an epitope comprised in residues 1-20, 20-40, 30-50, 60-80, 70-90,80-100, or 90-110. In another embodiment, the antagonist, optionally aproteinanceous antagonist, may bind an epitope comprised in the IgV,stalk region, cytoplasmic region or transmembrane region of said VISTAprotein. In another embodiment, the antagonist, optionally aproteinanceous antagonist, may elicit at least one of the followingactivities: (a) upregulates cytokines; (b) induces expansion of T cells,(c) promotes antigenic specific T cell immunity; or (d) promotes CD4+and/or CD8+ T cell activation.

In another embodiment, an isolated binding agent, preferably an antibodyor antibody fragment, may specifically binds to a VISTA (PD-L3) proteinmay comprise the amino acid sequence set forth in SEQ ID NO:2, 4 or 5 ora variant, fragment or ortholog thereof. In an embodiment, the bindingagent modulates (agonizes or antagonizes) VISTA activity in vitro or invivo. In one embodiment, the binding agent may be an agonistic orantagonistic anti-VISTA antibody.

In one embodiment, the anti-VISTA (PD-L3) antibodies may have functionalproperties including but not limited to modulating specific effects ofVISTA (PD-L3) on immunity such as the suppressive effect of the proteinon TCR activation, the suppressive effect of the protein on CD4 T cellproliferative responses to anti-CD3, suppression of antigen specificproliferative responses of cognate CD4 T cells, the suppressive effectsof VISTA (PD-L3) on the expression of specific cytokines (e.g., IL-2 andγ interferon).

In a further embodiment, antibodies, optionally monoclonal or polyclonalantibodies, may specifically bind VISTA (PD-L3) polypeptides includinghuman VISTA polypeptides.

In one embodiment, an isolated antibody, or antibody fragment thereof,that specifically binds to a VISTA polypeptide, multimeric VISTApolypeptide, or VISTA fusion protein. In another embodiment, theantibody or antibody fragment thereof may exhibit antitumor orantimetastatic activity. In another embodiment, the antibody or antibodyfragment thereof may specifically bind an epitope comprised in residues1-20, 20-40, 30-50, 60-80, 70-90, 80-100, or 90-110. In anotherembodiment, the antibody or antibody fragment thereof may specificallybind an epitope comprised in the IgV, stalk region, cytoplasmic regionor transmembrane region of said VISTA protein. In another embodiment,the antibody or antibody fragment thereof may elicit at least one of thefollowing activities: (a) upregulates cytokines; (b) induces expansionof T cells, (c) promotes antigenic specific T cell immunity; or (d)promotes CD4+ and/or CD8+ T cell activation. In another embodiment, theantibody or fragment may be recombinant. In another embodiment, theantibody or fragment may have anti-tumor activity. In anotherembodiment, the antibody fragment may be a Fab, Fab′, F(ab′)2, Fv, CDR,paratope, or portion of an antibody that is capable of binding theantigen. In another embodiment, the antibody may be chimeric, humanized,anti-idiotypic, single-chain, bifunctional, or co-specific. In anotherembodiment, the antibody or fragment may be directly or indirectlyconjugated to a label, cytotoxic agent, therapeutic agent, or animmunosuppressive agent. In a further embodiment, the may be achemiluminescent label, paramagnetic label, an MRI contrast agent,fluorescent label, bioluminescent label, or radioactive label.

In one embodiment, the invention provides anti-VISTA antibodies andantibody fragments thereof. In one embodiment, the antibody fragment isa Fab, Fab′, F(ab′)₂, Fv and scFv fragment. In one embodiment, theantibody or antibody fragment thereof may comprise a Fab, Fab′, F(ab′)₂,Fv, single-chain variable fragment (scFv), IgNAR, SMIP, camelbody, ornanobody. In another embodiment, a recombinant protein may comprise thehypervariable region of an anti-VISTA antibody and selectively bindVISTA. In another embodiment, the antibody fragment may selective bindVISTA may comprise the amino acid sequence of SEQ ID NO:2, 4 or 5.

In addition, the VISTA (PD-L3) polypeptides (or biologically activeportions thereof) or modulators of the VISTA (PD-L3) molecules (e.g.,anti-VISTA antibodies) may be incorporated into pharmaceuticalcompositions, optionally may comprise a pharmaceutically acceptablecarrier.

In another embodiment, the invention provides a vaccine may comprise anantigen and an agent that modulates (enhances or inhibits) VISTA (PD-L3)activity. In an embodiment, the vaccine inhibits the interaction betweenVISTA (PD-L3) and its natural binding partner(s). In another embodiment,a vaccine may comprise an antigen and an agent that inhibits theinteraction between VISTA (PD-L3) and its natural binding partner(s). Inanother embodiment, a vaccine may comprise an antigen and an agent thatpromotes the interaction between VISTA (PD-L3) and its natural bindingpartner(s). In one embodiment, the vaccine comprises an excipient,adjuvant, or a carrier.

In one embodiment, a kit may comprise a VISTA fusion protein. In anotherembodiment, a kit may comprise a multimeric VISTA protein. In a furtherembodiment, the VISTA fusion protein or multimeric VISTA protein may bedirectly or indirectly fixed to a solid phase support. In a furtherembodiment, the solid phase support may be a bead, test tube, sheet,culture dish, or test strip. In another embodiment the solid phasesupport may be an array.

In another embodiment, immune cells may be activated may comprisecontacting an immune cell with a VISTA polypeptide, VISTA-Ig fusionprotein, or anti-VISTA antibody. In another embodiment, the immune cellmay be a T cell, B cell, or an antigen-presenting cell. Immune cellsactivated in accordance with the method of the instant invention cansubsequently be expanded ex vivo and used in the treatment andprevention of a variety of diseases; e.g., human T cells which have beencloned and expanded in vitro maintain their regulatory activity. Priorto expansion, a source of T cells may be obtained from a subject (e.g.,a mammals such as a human, dog, cat, mouse, rat, or transgenic speciesthereof). T cells can be obtained from a number of sources, includingperipheral blood mononuclear cells, bone marrow, lymph node tissue, cordblood, thymus tissue, tissue from a site of infection, spleen tissue,tumors or T cell lines. T cells may be obtained from a unit of bloodcollected from a subject using any number of techniques known to theskilled artisan, such as FICOLL® separation.

In another embodiment, a method for modulating VISTA (PD-L3) activity,may comprise contacting a cell capable of expressing VISTA (PD-L3) withan agent that modulates VISTA (PD-L3) activity, preferably an anti-VISTA(PD-L3) antibody such that VISTA (PD-L3) activity in the cell may bemodulated. In one embodiment, the agent inhibits VISTA (PD-L3) activity.In another embodiment, the agent stimulates VISTA (PD-L3) activity. In afurther embodiment, the agent interferes with or enhances theinteraction between a VISTA (PD-L3) polypeptide and its natural bindingpartner(s). In one embodiment, the agent may be an antibody thatspecifically binds to a VISTA (PD-L3) polypeptide. In anotherembodiment, the agent may be a peptide, peptidomimetic, or other smallmolecule that binds to a VISTA (PD-L3) polypeptide.

In another embodiment, the agent modulates expression of VISTA (PD-L3)by modulating transcription of a VISTA (PD-L3) gene, translation of aVISTA (PD-L3) mRNA, or post-translational modification of a VISTA(PD-L3) polypeptide. In another embodiment, the agent may be a nucleicacid molecule having a nucleotide sequence that may be antisense to thecoding strand of a VISTA (PD-L3) mRNA or a VISTA (PD-L3) gene. In afurther embodiment, the agent may be an siRNA molecule that targetsVISTA (PD-L3) mRNA.

In one embodiment, methods for treating autoimmune disorder orinflammatory condition may comprise administering an agent which may bea VISTA (PD-L3) modulator to the subject. In one embodiment, the VISTA(PD-L3) modulator may be a VISTA (PD-L3) polypeptide, preferably asoluble fusion protein or multimeric VISTA protein or anti-VISTAantibody as described infra. In another embodiment the VISTA (PD-L3)modulator may be a VISTA (PD-L3) nucleic acid molecule, e.g., in anadenoviral vector. In another embodiment, the invention further providestreating the subject with an additional agent that modulates an immuneresponse.

In one embodiment, a method for modulating the interaction of VISTA(PD-L3) with its natural binding partner(s) on an immune cell maycomprise contacting an antigen presenting cell which expresses VISTA(PD-L3) with an agent selected from the group consisting of a form ofVISTA (PD-L3), or an agent that modulates the interaction of VISTA(PD-L3) and its natural binding partner(s) such that the interaction ofVISTA (PD-L3) with it natural binding partner(s) on an immune cell maybe modulated and assessing the interaction of VISTA with its naturalbinding partner(s). In an embodiment, an agent that modulates theinteraction of VISTA (PD-L3) and its natural binding partner(s) may bean antibody that specifically binds to VISTA (PD-L3). In one embodiment,the interaction of VISTA (PD-L3) with its natural binding partner(s) maybe upregulated. In another embodiment, the interaction of VISTA (PD-L3)with its natural binding partner(s) may be downregulated. In oneembodiment, the method further comprises contacting the immune cell orthe antigen presenting cell with an additional agent that modulates animmune response. In one embodiment, the step of contacting may beperformed in vitro. In another embodiment, the step of contacting may beperformed in vivo. In one embodiment, the immune cell may be selectedfrom the group consisting of a T cell, a monocyte, a macrophage, adendritic cell, a B cell, and a myeloid cell.

In one embodiment, a method for inhibiting activation in an immune cellmay comprise inhibiting the activity or expression of VISTA (PD-L3) in acell such that immune cell activation may be inhibited. In oneembodiment, a method for increasing activation in an immune cell maycomprise increasing the activity or expression of VISTA (PD-L3) in acell such that immune cell activation may be increased.

In another embodiment, a method for upregulating an immune response maycomprise administering an agent that inhibits the interaction betweenVISTA (PD-L3) and its natural binding partner(s) on immune cells. In oneembodiment, the agent comprises a blocking antibody or a small moleculethat binds to VISTA (PD-L3) and inhibits the interaction between VISTA(PD-L3) and its natural binding partner(s). In another embodiment, themethod further comprises administering a second agent that upregulatesan immune response to the subject. In another embodiment, a method fordownregulating an immune response may comprise administering an agentthat stimulates the interaction between VISTA (PD-L3) and its naturalbinding partner(s) on immune cells.

In one embodiment, a method for treating a condition selected from thegroup consisting of a tumor, a pathogenic infection, an inflammatoryimmune response or condition, preferably less pronounced inflammatoryconditions, or an immunosuppressive disease may comprise administrationof an effective amount of a VISTA polypeptide or VISTA-Ig fusionprotein. Specific examples include multiple sclerosis, thyroiditis,rheumatoid arthritis, diabetes type II and type I and cancers, bothadvanced and early forms, including metastatic cancers (e.g., bladdercancer, ovarian cancer, melanoma, lung cancer), wherein VISTA suppressesan effective anti-tumor response. The subject may be administered cellsor a viral vector that express a nucleic acid that encodes an anti-VISTAantibody or VISTA fusion protein.

In one embodiment, a method for treating a condition selected from thegroup consisting of transplant, an allergy, infectious disease, cancer,and inflammatory or autoimmune disorders (e.g., an inflammatory immunedisorder) may comprise administration of an effective amount of a VISTA(PD-L3) proteins, binding agents or VISTA (PD-L3) antagonists oragonists. In another embodiment, type 1 diabetes, multiple sclerosis,rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus,rheumatic diseases, allergic disorders, asthma, allergic rhinitis, skindisorders, gastrointestinal disorders such as Crohn's disease andulcerative colitis, transplant rejection, poststreptococcal andautoimmune renal failure, septic shock, systemic inflammatory responsesyndrome (SIRS), adult respiratory distress syndrome (ARDS) andenvenomation; autoinflammatory diseases as well as degenerative bone andjoint diseases including osteoarthritis, crystal arthritis andcapsulitis and other arthropathies may be treated may compriseadministration of an effective amount of a VISTA (PD-L3) proteins,binding agents or VISTA (PD-L3) antagonists or agonists. Further, themethods and compositions may comprise an effective amount of a VISTA(PD-L3) proteins, binding agents or VISTA (PD-L3) antagonists oragonists may be used for treating tendonitis, ligamentitis and traumaticjoint injury. In one embodiment, an agent comprises an antibody or asmall molecule that stimulates the interaction between VISTA (PD-L3) andits natural binding partner(s). In another embodiment, the methodfurther comprises administering a second agent that downregulates animmune response to the subject such as a PD-L1, PD-L2 or CTLA-4 fusionprotein or antibody specific thereto.

In embodiments the subject VISTA (PD-L3) proteins, nucleic acids, andligands specific to VISTA (PD-L3), preferably antibodies having desiredeffects on VISTA (PD-L3) functions may be used to treat conditionsincluding but not limited to cancer, autoimmune diseases, allergy,inflammatory disorders or infection and more specifically immune systemdisorders such as severe combined immunodeficiency, multiple sclerosis,systemic lupus erythematosus, type I diabetes mellitus,lymphoproliferative syndrome, inflammatory bowel disease, allergies,asthma, graft-versus-host disease, and transplant rejection; immuneresponses to infectious pathogens such as bacteria and viruses; andimmune system cancers such as lymphomas and leukemias. In oneembodiment, an agent that modulates the activity of VISTA may relieve Tcell exhaustion and enhance immunity to infectious disease.

In one embodiment, a method of treating a cancer in a patient in needthereof may comprise administering an effective amount of VISTA protein,multimeric VISTA protein, VISTA fusion protein, optionally a VISTA-Igfusion protein, wherein said VISTA protein, multimeric VISTA protein,and/or VISTA fusion protein enhances antitumor immunity by suppressingthe immunosuppressive activity of VISTA expressed by myeloid dendriticsuppressor cells. In a further embodiment, the patient prior totreatment may be found to express elevated levels of VISTA protein onimmune cells.

In one embodiment, a method of enhancing the efficacy of radiotherapy,chemotherapy or an anti-cancer biologic may comprise administering aneffective amount of VISTA protein, multimeric VISTA protein, VISTAfusion protein, optionally a VISTA-Ig fusion protein, in a therapeuticregimen including the administration of radiotherapy, chemotherapy or ananti-cancer biologic. In a further embodiment, the patient prior totreatment may have a cancer that does not respond to said radiotherapy,chemotherapy or an anti-cancer biologic.

In one embodiment, a method of treating colorectal, bladder, ovarian, ormelanoma cancer may comprise administering an effective amount of VISTAprotein, multimeric VISTA protein, VISTA fusion protein, optionally aVISTA-Ig fusion protein, wherein said cancer is in early(non-metastatic) or metastatic form and the VISTA-Ig blocks interactionwith its receptor.

In one embodiment, a method for modulating an immune cell response maycomprise contacting an immune cell with an effective amount of a VISTAprotein, multimeric VISTA protein, VISTA fusion protein, optionally aVISTA-Ig fusion protein in the presence of a primary signal so that aresponse of the immune cell is modulated.

In one embodiment, a method of modulating Treg cells in a subject inneed thereof may comprise administering an effective amount of VISTAprotein, multimeric VISTA protein, VISTA fusion protein, optionally aVISTA-Ig fusion protein.

In one embodiment, a method of releasing the suppressive effect of VISTAon immunity may comprise administering an effective amount of a VISTAprotein, multimeric VISTA protein, VISTA fusion protein, optionally aVISTA-Ig fusion protein. In another embodiment, the treated patient maybe found to express elevated levels of VISTA prior to treatment. Inanother embodiment, the VISTA levels may be monitored after treatment inorder to assess that the immune response may have been enhanced.

In one embodiment, a method of enhancing cell mediated immunity in asubject in need thereof may comprise administering an effective amountof a VISTA protein, multimeric VISTA protein, VISTA fusion protein,optionally a VISTA-Ig fusion protein.

In one embodiment, a method for modulating an immune cell response maycomprise contacting an immune cell with may comprise administering aneffective amount of a VISTA fusion protein, optionally a VISTA-Ig fusionprotein, or a multimeric VISTA protein in the presence of a primarysignal so that a response of the immune cell is modulated. In anotherembodiment, the contacting may be performed in vitro, in vivo, or exvivo.

In one embodiment, a method of regulating T cell responses duringcognate interactions between T cells and myeloid derived APCs maycomprise administering an effective amount of a VISTA fusion protein,optionally a VISTA-Ig fusion protein, or a multimeric VISTA protein.

In one embodiment, a method of eliciting immunosuppression in anindividual in need thereof may comprise administering an effectiveamount of a VISTA fusion protein, optionally a VISTA-Ig fusion protein,or a multimeric VISTA protein.

In another embodiment, a method for decreasing immune cell activationmay comprise administering an effective amount of a VISTA (PD-L3)polypeptide or VISTA-Ig fusion protein to a subject, wherein said VISTA(PD-L3) polypeptide or VISTA-Ig fusion protein acts as inhibitory signalfor decreasing immune cell activation. In one embodiment, the immunecell activation is inhibited. In another embodiment, the immune cellactivation is significantly decreased. In one embodiment, the inhibitorysignal binds to an inhibitory receptor (e.g., CTLA-4 or PD-1) on animmune cell thereby antagonizing the primary signal which binds to anactivating receptor (e.g., via a TCR, CD3, BCR, or Fc polypeptide). Inone embodiment, the VISTA polypeptide or VISTA-Ig fusion proteininhibits second messenger generation; inhibits immune cellproliferation; inhibits effector function in the immune cell (e.g.,reduced phagocytosis, reduced antibody production, reduced cellularcytotoxicity, the failure of the immune cell to produce mediators,(cytokines (e.g., IL-2) and/or mediators of allergic responses); or thedevelopment of anergy.)

In one embodiment, the primary signal may be a ligand (e.g., CD3 oranti-CD3) that binds TCR and initiates a primary stimulation signal. TCRligands include but are not limited to anti-CD3 antibody OKT3 andanti-CD3 monoclonal antibody G19-4. In one embodiment, a primary signalmay be delivered to a T cell through other mechanisms including aprotein kinase C activator, such as a phorbol ester (e.g., phorbolmyristate acetate), and a calcium ionophore (e.g., ionomycin, whichraises cytoplasmic calcium concentrations). The use of such agentsbypasses the TCR/CD3 complex but delivers a stimulatory signal to Tcells. Other agents acting as primary signals may include natural andsynthetic ligands. A natural ligand may comprise MHC with or without apeptide presented. Other ligands may include, but are not limited to, apeptide, polypeptide, growth factor, cytokine, chemokine, glycopeptide,soluble receptor, steroid, hormone, mitogen (e.g., PHA), or othersuperantigens, peptide-MHC tetramers and soluble MHC dimers.

In another embodiment, a method for detecting the presence of a VISTA(PD-L3) nucleic acid molecule, protein, or polypeptide in a biologicalsample comprises contacting the biological sample with an agent capableof detecting a VISTA (PD-L3) nucleic acid molecule, protein, orpolypeptide, such that the presence of a VISTA (PD-L3) nucleic acidmolecule, protein or polypeptide may be detected in the biologicalsample. This VISTA (PD-L3) expression may be used to detect certaindisease sites such as inflammatory sites.

In another embodiment, a method for detecting the presence of VISTA(PD-L3) activity in a biological sample comprises contacting thebiological sample with an agent capable of detecting an indicator ofVISTA (PD-L3) activity, such that the presence of VISTA (PD-L3) activitymay be detected in the biological sample. In a further embodiment, amethod for detecting soluble VISTA in biological sample may comprisecontacting the biological sample with an agent capable of detecting anindicator of VISTA (PD-L3) activity, such that the presence of VISTA(PD-L3) activity may be detected in the biological sample. In anotherembodiment, a method for detecting soluble VISTA in biological samplemay comprise contacting the biological sample with an agent capable ofbinding VISTA (PD-L3), optionally an anti-VISTA antibody or antibodyfragment, and detecting the presence of VISTA-antibody complexes. In afurther embodiment, the measurement may be quantitative, optionallyWestern blot densitometry, colorimetric, or fluorometic.

In another embodiment, diagnostic assays for identifying the presence orabsence of a genetic alteration in a VISTA gene comprises obtaining asample may comprise a nucleic acid and analyzing the sample, whereinsaid genetic alteration is characterized by at least one of (i) aberrantmodification or mutation of a gene encoding a VISTA (PD-L3) polypeptide;(ii) misregulation of the gene; and (iii) aberrant post-translationalmodification of a VISTA (PD-L3) polypeptide, wherein a wild-type form ofthe gene encodes a polypeptide with a VISTA (PD-L3) activity. In oneembodiment, the nucleic acid may be DNA or mRNA.

In one embodiment, a method of selecting for anti-VISTA antibodies forhaving potential use as a therapeutic or immune modulatory agent maycomprise: (a) immunizing immune cells or a host with a VISTA protein,immunogenic fragment, or conjugate thereof; (b) selecting lymphoid cellswhich express antibodies that specifically bind to VISTA; (c) selectinganti-VISTA antibodies or antibody fragments thereof; (d) screening saidanti-VISTA antibodies or antibody fragments thereof for the ability toinhibit or enhance at least one of the following activities of VISTA(PD-L3) or VISTA: (i) suppression of T cell activation ordifferentiation; (ii) suppression of CD4+ or CD8+ T cell proliferation,or suppression of cytokine production by T cells; (iii) wherein anantibody or antibody fragment thereof which has at least one of theactivities in (d) has potential use as a therapeutic or immunemodulatory agents.

In further embodiment, methods of selecting anti-VISTA (PD-L3)antibodies having desired functional properties may comprise screeningpanels of monoclonal antibodies produced against this protein or a VISTA(PD-L3)-Ig fusion protein based on desired functional propertiesincluding modulating specific effects of VISTA (PD-L3) on immunity suchas the suppressive effect of the protein on TCR activation, thesuppressive effect of the protein on CD4 T cell proliferative responsesto anti-CD3, suppression of antigen specific proliferative responses ofcognate CD4 T cells, the suppressive effects of VISTA (PD-L3) on theexpression of specific cytokines (e.g., IL-2 and γ interferon) andselecting the desired antibody.

In another embodiment, methods for identifying a compound that binds toor modulates the activity of a VISTA (PD-L3) polypeptide may compriseproviding an indicator composition may comprise a VISTA (PD-L3)polypeptide having VISTA (PD-L3) activity, contacting the indicatorcomposition with a test compound, and determining the effect of the testcompound on VISTA (PD-L3) activity in the indicator composition toidentify a compound that modulates the activity of a VISTA (PD-L3)polypeptide.

In another embodiment, a cell-based assay for screening for compoundswhich modulate the activity of VISTA (PD-L3) may comprise contacting acell expressing a VISTA (PD-L3) target molecule with a test compound anddetermining the ability of the test compound to modulate the activity ofthe VISTA (PD-L3) target molecule

In another embodiment, a cell-free assay for screening for compoundswhich modulate the binding of VISTA (PD-L3) to a target molecule maycomprise contacting a VISTA (PD-L3) polypeptide or biologically activeportion thereof with a test compound and determining the ability of thetest compound to bind to the VISTA (PD-L3) polypeptide or biologicallyactive portion thereof.

In another embodiment, a method of identifying a compound, e.g. ananti-VISTA (PD-L3) antibody which modulates the effect of VISTA (PD-L3)on T cell activation or cytokine production at a first and secondantigen concentration may comprise contacting a T cell expressing aVISTA (PD-L3) target molecule with a test compound at a first antigenconcentration, determining the ability of the test compound to modulateT cell proliferation or cytokine production at the first antigenconcentration, contacting a T cell expressing a VISTA (PD-L3) targetmolecule with the test compound at a second antigen concentration, anddetermining the ability of the test compound to modulate T cellproliferation or cytokine production at the second antigenconcentration, thereby identifying a compound which modulates T cellactivation or cytokine production at a first and second antigenconcentration.

In other embodiments panels of anti-VISTA (PD-L3) antibodies and VISTA(PD-L3) proteins may be screened and selected on the basis of whichanti-VISTA antibodies inhibit or promote the effects of VISTA (PD-L3) onCD4+ and CD8+ T cell differentiation, proliferation and/or cytokineproduction. In a further embodiment, a mouse that has been engineered toexpress human VISTA may be used to test the function of anti-human VISTAantibodies in regulating immunity.

In another embodiment, a method of treating graft-versus-host-disease(GVHD) may comprise administration of an effective amount of a VISTAfusion protein, optionally a VISTA-Ig fusion protein, or the multimericVISTA protein. In another embodiment, a method for treatinggraft-versus-host disease (GVHD), acute graft-versus-host disease,chronic graft-versus-host disease, acute graft-versus-host diseaseassociated with stem cell transplant, chronic graft-versus-host diseaseassociated with stem cell transplant, acute graft-versus-host diseaseassociated with bone marrow transplant, acute graft-versus-host diseaseassociated with allogeneic hemapoetic stem cell transplant (HSCT), orchronic graft-versus-host disease associated with bone marrow transplantmay comprise administering of an effective amount of a VISTA fusionprotein, optionally a VISTA-Ig fusion protein, or the multimeric VISTAprotein.

In one embodiment, the graft-versus-host disease (GVHD) may begraft-versus-host disease (GVHD), acute graft-versus-host disease,chronic graft-versus-host disease, acute graft-versus-host diseaseassociated with stem cell transplant, chronic graft-versus-host diseaseassociated with stem cell transplant, acute graft-versus-host diseaseassociated with bone marrow transplant, acute graft-versus-host diseaseassociated with allogeneic hemapoetic stem cell transplant (HSCT), orchronic graft-versus-host disease associated with bone marrowtransplant. In another embodiment, the patient treated has at least onesymptom of graft-versus-host disease (GVHD), optionally wherein thepatient exhibits acute GVHD includes but is not limited to abdominalpain, abdominal cramps, diarrhea, fever, jaundice, skin rash, vomiting,and weight loss. In another embodiment, the patient treated has at leastone symptom of chronic graft-versus-host disease (GVHD) includes but isnot limited to dry eyes, dry mouth, hair loss, hepatisis, lung disorder,gastrointestinal tract disorders, skin rash, and skin thickening. Inanother embodiment, the patient has or is to receive allogeneic stemcell or bone marrow transplant. In another embodiment, the patient hasor is to receive autologous stem cell or bone marrow transplant.

DETAILED DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E depict sequence analysis. (A) Full length amino acidsequence of murine VISTA (PD-L3) (SEQ ID NO: 17). (B) Amino acidsequence alignment of extracellular Ig domains between murine VISTA(PD-L3) (SEQ ID NO: 25) and selected B7 family ligands, including B7-H1(PD-L1) (SEQ ID NO: 26), B7-DC (PD-L2) (SEQ ID NO: 27), B7-H3 (CD276)(SEQ ID NO: 28), and B7-H4 (B7S1) (SEQ ID NO: 29). (C) Alignment ofVISTA (PD-L3) (SEQ ID NO: 30) Ig domain with B7 family receptors,including PD-1 (SEQ ID NO: 31), CTLA-4 (SEQ ID NO: 32), CD28 (SEQ ID NO:33), BTLA (SEQ ID NO: 34), and ICOS (SEQ ID NO: 35). Ig-v domain, “ . .. ”; Ig-c domain, “_”. Alignment was performed using the MUSCLEalgorithm (Multiple Sequence Comparison by Log-Expectation). (D)Sequence identity (%) of the Ig-V domains between VISTA (PD-L3) andother B7 family ligands and receptors is calculated using ClustalW2program. (E) Sequence alignment to show sequence homology between human(SEQ ID NO: 37) and murine VISTA (PD-L3) (SEQ ID NO: 36). Identicalresidues are shaded in black. Highly conserved and semi-conservedresidues are shaded in dark and light shade of gray respectively.

FIG. 2 depicts a hylogenic analysis of mouse VISTA (PD-L3) with otherImmunoglobulin (Ig) superfamily members. Full-length sequence of mouseVISTA (PD-L3) and other Ig superfamily members, including CD28, CTLA-4,ICOS, BTLA, PD-1, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2, B7-H3, B7-H4,B7-1, B7-2, BTNL2, BTN3A3, BTN2A2, and BTN1A1, were analyzed using PhyMLalgorithm (Phylogenetic Maximum Likelihood). Branch distances were shownat tree branch joints.

FIGS. 3A-G depict the tissue expression and hematopoietic cellexpression patterns of VISTA (PD-L3) A. RT-PCR of full length VISTA(PD-L3) from mouse tissues. Lanes: (1)muscle (2)heart (3)eye (4) thymus(5)spleen (6)small intestine (7)kidney (8)liver (9)brain (10)mammarygland (11)lung (12)ovary (13)bone marrow. B. RT-PCR of full-length VISTA(PD-L3) from purified hematopoietic cell types. Lanes (1) peritonealmacrophages (2) splenic CD11b+ monocytes (3) splenic CD11c+ DCs (4)splenic CD4+ T cells (5) splenic CD8+ T cells (6) splenic B cells. C-E.Flow cytometry analysis of VISTA (PD-L3) expression on splenic CD4+ andCD8+ T cells from thymus and spleen (C), on CD11b+ monocytes (D), and onCD11c+DC subsets from spleen and peritoneal cavity (E). (F) Splenic Bcells, NK cells and granulocytes are also analyzed. (G) The differentialexpression of VISTA (PD-L3) on hematopoietic cells from different tissuesites, including mesenteric LN, peripheral LN, spleen, blood andperitoneal cavity. Representative data from at least 3 independentexperiments are shown.

FIG. 4 depicts a VISTA, novel and structurally-distinct, Ig-superfamilyinhibitory ligand, whose extracellular domain bears highest homology tothe B7 family ligand PD-L1 as displayed on an Antigen Presenting Cellalong with other CDs and B7 family members. VISTA has a 93 aacytoplasmic domain with no obvious signal transducing motifs, except apossible protein kinase C binding site.

FIG. 5 depicts the specificity of VISTA (PD-L3) hamster monoclonalantibodies. Mouse EL4 cell lines over-expressing either PD-L1 or VISTA(PD-L3) fused to RFP were stained using the supernatants from hybridomacultures and analyzed by flow cytometry. Two representative positiveclones are shown, 8D8 AND 6E7.

FIG. 6 depicts a comparison of VISTA (PD-L3) expression with other B7family ligands on in vitro cultured spleen cells. Expression of VISTA(PD-L3) and other B7 family ligands (i.e., PD-L1, PD-L2, B7-H3, andB7-H4) on hematopoietic cell types, including CD4+ T cells, CD11bhimonocytes, and CD11c+ DCs were compared. Cells were either freshlyisolated, or in vitro cultured for 24 hrs, with and without activation.CD4+ T cells were activated with plate-bound αCD3 (5 μg/ml), CD11bhimonocytes and CD11c+ DCs were activated with IFNα (20 ng/ml) and LPS(200 ng/ml). Representative results from three independent experimentsare shown.

FIGS. 7A-7B depict the comparison of in vivo expression patterns ofVISTA (PD-L3) and other B7 family ligands during immunization. DO11.10TCR transgenic mice were immunized with chicken ovalbumin (OVA)emulsified in complete Freund's adjuvant (CFA) on the flank. Drainingand non-draining lymph node cells were collected 24 hr postimmunization, and analyzed by flow cytometry for the expression of VISTA(PD-L3), PD-L1 and PD-L2. Shown are representative results from at leastfour independent experiments. (A) A population of CD11b+ cellsexpressing a high level of VISTA (PD-L3) was induced at 24 hr postimmunization with CFA/OVA, but not with CFA alone within the draininglymph node. These cells are of mixed phenotype of F4/80+ macrophages andCD11C+ dendritic cells. (B) Expression of VISTA (PD-L3), PD-L1 and PD-L2on CD11bhi monocytes, CD11c+ DCs and CD4+ T cells were analyzed at 24 hrpost immunization.

FIG. 8 depicts the loss of VISTA (PD-L3) expression on activated CD4+ Tcells, CD11b⁺ and CD11c⁺ cells in response to immunization. DO11.10 micewere immunized with chicken ovalbumin (OVA) emulsified in completeFreund's adjuvant (CFA) on the flank. Draining and non-draining lymphnode cells were collected 48 hr post immunization, and analyzed forVISTA (PD-L3) expression by flow cytometry. Shown are representativeresults from 2 independent experiments.

FIGS. 9A-9D depict that immobilized VISTA (PD-L3)-Ig fusion proteininhibited CD4+ and CD8+ T cell proliferation. (A) CFSE labeled CD4+ andCD8+ T cells were stimulated by plate-bound αCD3 with or withoutco-absorbed VISTA (PD-L3)-Ig. The percentage of CFSE-low cells wasquantified and shown in (B). (C) CD4+ T cells from PD-1 ko mice werealso suppressed by VISTA (PD-L3)-Ig. (D) VISTA (PD-L3)-Ig-mediatedsuppression is persistent and can act late. CD4+ T cells were activatedin the presence of VISTA (PD-L3)-Ig or control-Ig for either 72 hrs (i),or for 24 hrs (ii, iii and iv). 24 hour-preactivated cells wereharvested and re-stimulated under specified conditions for another 48hours. Cell proliferation was analyzed at the end of the 72 hourculture. (ii) Pre-activation with VISTA (PD-L3)-Ig and re-stimulationwith antiCD3; (iii) Pre-activation with antiCD3 and re-stimulation withVISTA (PD-L3)-Ig. (iv) Pre-activation with VISTA (PD-L3)-Ig andre-stimulation with VISTA (PD-L3)-Ig. Duplicated wells were analyzed forall conditions. Shown are representative results from four experiments.

FIG. 10 depicts the similar inhibitory effect of PD-L1-Ig and VISTA(PD-L3)-Ig fusion proteins on CD4+ T cell proliferation. Bulk purifiedCD4+ T cells were CFSE labeled and stimulated with plate-bound αCD3together with titrated amount of PD-L1-Ig or VISTA (PD-L3)-Ig fusionproteins. CFSE dilution was analyzed at 72 hours and the percentage ofCFSElow cells was quantified. Duplicated wells were analyzed for allconditions. Shown are representative results from 2 independentexperiments.

FIGS. 11A-B depict the suppressive impact of VISTA (PD-L3)-Ig on theproliferation of nave and memory CD4+ T cells. (A) Nave(CD25-CD44lowCD62Lhi) and memory (CD25-CD44hiCD62Llow) CD4+ T cellsubsets were sorted, CFSE labeled, and stimulated with plate-boundanti-CD3 (2.5 μg/ml) together with VISTA (PD-L3)-Ig or control-Ig atindicated ratios. Cell proliferation was analyzed at 72 hours byexamining the CFSE division profile. The percentage of proliferatedcells, as determined by percentage of CFSElow cells, is calculated andshown in B. Duplicated wells were analyzed for all conditions. Shown arerepresentative results from two independent experiments.

FIGS. 12A-12B depict VISTA (PD-L3)-Ig fusion protein suppressed earlyTCR activation and cell proliferation, but did not directly induceapoptosis. Bulk purified CD4+ T cells were stimulated with plate-boundanti-CD3 together with VISTA (PD-L3)-Ig or control-Ig at 1-2 ratio (2.5μg/ml and 5 μg/ml respectively). Cells were analyzed at 24 hr and 48 hrsfor the expression of CD69, CD62L, and CD44 by flow cytometry. Cellswere also stained for early apoptosis marker annexin-V, and cell deathmarker 7-Aminoactinomycin D (7-AAD). Shown are representative resultsfrom two independent experiments.

FIGS. 13A-13E depict VISTA-Ig inhibited cytokine production by CD4+ andCD8+ T cells. (A-B) Bulk purified CD4+ T cells were stimulated withplate-bound anti-CD3, and VISTA-Ig or control-Ig at stated ratios.Culture supernatants were collected after 24 hrs and 48 hrs. Levels ofIL-2 and IFNγ were analyzed by ELISA. (C-D) CD4+ T cells were sortedinto naïve (CD25-CD44lowCD62Lhi) and memory (CD25-CD44hiCD62Llow) cellpopulations. Cells were stimulated with plate-bound αCD3 and VISTA(PD-L3)-Ig or control-Ig at a ratio of 1:2. Culture supernatants werecollected at 48 hrs and analyzed for the level of IL-2 and IFN γ byELISA. (E) Bulk purified CD8+ T cells were stimulated with plate-boundαCD3, and VISTA (PD-L3)-Ig or control-Ig at indicated ratios. IFN γ inthe culture supernatant was analyzed by ELISA. For all conditions,supernatant for six duplicated wells were pooled for ELISA analysis.Shown are representative results from three experiments.

FIGS. 14A-14D depict VISTA-Ig-mediated suppression may overcome amoderate level of costimulation provided by CD28, but was completelyreversed by a high level of costimulation, as well as partially rescuedby exogenous IL-2. A-B. Mouse CD4+ T cells were activated by plate-boundαCD3 together with either VISTA (PD-L3)-Ig or control-Ig at 1-1 ratioand 1-2 ratios. For cytokine rescue, soluble mIL-2, mIL7, mIL15 andmIL-23 (all at 40 ng/ml) were added to the cell culture (A). To examinethe effects of costimulation, αCD28 (1 μg/ml) was immobilized togetherwith αCD3 and Ig proteins at indicated ratios (B). Cell proliferationwas analyzed at 72 hr by examining CFSE division profiles. C-D. Toexamine the suppressive activity of VISTA (PD-L3) in the presence oflower levels of costimulation, titrated amounts of αCD28 were coatedtogether with anti-CD3 (2.5 μg/ml) and VISTA-Ig fusion proteins orcontrol-Ig fusion protein (10 μg/ml) to stimulate mouse CD4+ T cellproliferation. Cell proliferation was analyzed at 72 hour. Percentagesof proliferated CFSElow cells were quantified and shown in D. Duplicatedwells were analyzed for all conditions. Representative CFSE profilesfrom three independent experiments are shown.

FIGS. 15A-15D depict that VISTA (PD-L3) expressed on antigen presentingcells suppressed CD4 T cell proliferation. A-C The CHO cell line thatstably expresses MHCII molecule I-Ad and costimulation molecule B7-2 wasused as the parent cell line. Cells were transduced with retrovirusexpressing either VISTA-RFP or RFP control molecules. Transduced cellswere sorted to achieve homogenous level of expression. To test theirability as antigen presenting cells, CHO-VISTA or CHO-RFP cells weremitomycin C treated and mixed with OVA-specific transgenic CD4+ T cellsD011.10, in the presence of titrated amount of OVA peptide.Proliferation of D011 cells was analyzed at 72 hrs, either by CFSEdivision profiles (A-B), or by tritium incorporation (C). (D) bonemarrow derived dendritic cells were transduced with RFP or B7B-H5-RFPretrovirus during 10-day culture period. Transduced CD11c+ RFP+ DCs andnon-transduced CD11c+ RFP− DCs were sorted and used to stimulateOVA-specific transgenic CD4+ T cells OTII in the presence of titratedamount of OVA peptide. Cell proliferation was analyzed on day 3 byexamining CFSE division. For all experiments, duplicated wells wereanalyzed for all conditions, and representative results from threeindependent experiments are shown.

FIG. 16 depicts the surface expression level of VISTA (PD-L3) inretrovirally transduced bone marrow derived DCs. Bone marrow derived DCs(BMDC) were cultured in the presence of GM-CSF (20 ng/mml) andtransduced with either RFP or VISTA-RFP retrovirus as described herein.On day 10, surface expression level of VISTA were analyzed on culturedBMDCs, and compared to freshly-isolated peritoneal macrophages.

FIG. 17 shows that anti-PDL3 monoclonal antibody exhibits efficacy in apassive transfer EAE model. In this adoptive transfer EAE model, donorSJL mice were immunized with CFA and PLP peptide. On day 10, totallymphocytes from draining LN were isolated, and cultured in vitro withPLP peptide, IL-23 (20 ng/ml) and anti-IFNg (10 μg/ml) for 4 days.Expanded CD4 T cells were then purified and adoptively transferred intonaïve recipient mice. Disease progression was monitored and scored with:0, no disease; 0.5 loss of tail tone; 1: limp tail; 2: limp tail+hindlimb paresis; 2.5: 1 hind limb paralysis; 3: both hind limb paralysis;3.5: forelimb weakness; 4: hind limb paralysis+unilateral forelimbparalysis. Mice were sacrificed when disease score reached 4. *, micewere sacrificed.

FIG. 18 shows that VISTA expressed on antigen-presenting cellssuppressed CD4+ T cell proliferation.

FIG. 19 shows that an anti-VISTA antibody inhibited tumor growth in micetransplanted with MB49 tumor cells.

FIGS. 20A-20E show the antitumor effect of VISTA monoclonal antibodiesin four different mouse anti-tumor models (A-D). FIG. 21E shows theexpression of VISTA on different cells in the ID8 model. Very highexpression on the myeloid dendritic cells in different anatomiclocations. As can be seen, very high levels on myeloid dendritic cellsin the ascites cells, the site where the tumor grows and leukocytesinfiltrate.

FIG. 21 shows the potentiating effect of VISTA monoclonal antibodies onthe efficacy of a CD40/TLR agonist vaccine (consisting of using anagonistic αCD40 mab, TLR agonist and OVA peptide).

FIG. 22 shows VISTA expression on CNS cells in mice that are healthy orin mice that are developing EAE.

FIGS. 23A-23C depict a sequence and structural analysis of VISTA. (A)The primary amino acid sequence of mouse VISTA with the Ig-V domain, thestalk segment, and the transmembrane region highlighted in bold,italics, and Times New Roman, respectively. Cysteines in the ectodomainregion are indicated by underlining. (B) Multiple sequence alignment ofthe Ig-V domains of several B7 family members and VISTA. The predictedsecondary structure (using arrows, springs, and “T”s for strands,helices, and B-turns, respectively) is marked above the alignment and isbased on the VISTA structural model. VISTA (SEQ ID NO: 15), PD1L1 (SEQID NO: 11), PD1L2 (SEQ ID NO: 12), B7H4 (SEQ ID NO: 13), and B7H3 (SEQID NO: 14). (C) Multiple sequence alignment of VISTA orthologues.Invariant residues are represented by the red background, andphysico-chemically conserved positions are represented by red letters.Conserved amino acids are marked by blue boxes. Conservation iscalculated on the basis of 36 VISTA orthologous proteins, but only 9representatives are shown. The canonical cysteine pair (within the “B”and “F” strands) that is conserved in almost all Ig superfamily membersis highlighted by red circles, whereas cysteines that are specific toVISTA are marked by blue circles. The unique VISTA cysteine pattern isconserved in all orthologues from mouse (SEQ ID NO: 17), human (SEQ IDNO: 16), kangaroo (SEQ ID NO: 18), dolphin (SEQ ID NO: 19), chicken (SEQID NO: 20), xenopus (SEQ ID NO: 21), zebra finch (SEQ ID NO: 22),zebrafish, and fugu (SEQ ID NO: 23).

FIGS. 24A-24B depict that VISTA over expression on tumor cells overcomesprotective antitumor immunity. MCA105 tumor cells over expressing VISTAor RFP control protein were generated by retroviral transduction andsorted to homogeneity. To generate protective immunity, naive mice werevaccinated with irradiated MCA105 tumor cells subcutaneously on the leftflank. (A) Vaccinated mice were challenged 14 day later with liveMCA105VISTA or MCA105RFP tumor cells subcutaneously on the right flank.Tumor growth was monitored every 2 d. Tumor size is shown as mean±SEM.Shown are representative results from three independent repeats. (B)Vaccinated mice were either untreated or depleted of both CD4⁺ and CD8⁺T cells by monoclonal antibodies before live tumor challenge. Tumor sizewas monitored as in A and shown as mean±SEM. Shown are representativeresults from two independent repeats. For all experiments, ratiosindicate the number of tumor-bearing mice among total number of mice pergroup. The statistical differences (p-values) were assessed with anunpaired Mann-Whitney test.

FIG. 25A-D depicts that VISTA blockade using a specific monoclonalantibody enhanced CD4⁺ T cell response in vitro and in vivo. (A) Amonoclonal antibody clone 13F3 neutralized VISTA-mediated suppression invitro. A20-RFP and A20-VISTA cells were used to stimulate CFSE-labeledDO11.10 CD4⁺ T cells in the presence of cognate OVA peptide. 20 μg/mlVISTA-specific monoclonal antibody 13F3 or control-Ig was added asindicated. CFSE dilution was analyzed after 72 h, and percentages ofCFSE^(low) cells are shown as mean±SEM. Duplicated wells were analyzedfor all conditions. (B and C) Total CD11b^(hi) myeloid cells (B) orCD11b-CD11c⁻ monocytes (C) and CD11b^(hi)CD11c⁺ myeloid DCs (D) sortedfrom naive splenocytes were irradiated and used to stimulateCFSE-labeled OT-II transgenic CD4⁺ T cells in the presence of OVApeptide. Cell proliferation was measured by incorporation of tritiatedthymidine during the last 8 h of a 72-h culture period and shown asmean±SEM. Triplicate wells were analyzed in all conditions.

FIG. 26 depicts VISTA-IgG2a reduces Experimental AutoimmuneEncephalomyelitis (EAE) (a model of multiple sclerosis) progression.Mice were immunized with 175 μg MOG/CFA and pertussis toxin (PT) 300 ng(day 0, 2) to induce active EAE. On day 14, 17, and 20, 150 μg VISTA-IgG2a (n=8) or 150 μg control IgG2a (n=8) was administered. The data isshown as the mean±SEM.

FIG. 27 depicts the therapeutic effect of VISTA-IgG1 and VISTA-IgG2a onExperimental Autoimmune Encephalomyelitis (EAE) progression. Mice wereimmunized with 175 μg MOG/CFA and pertussis toxin (PT) 300 ng (day 0, 2)to induce active EAE. On day 6, mice were treated with 3 doses per weekof 150 μg control IgG1 (n=3), 150 μg control IgG2a (n=6), 150 μgmVISTA-IgG1 (n=3), or 150 μg mVISTA IgG2a (n=6) (two weeks in total).The data is shown as the mean±SEM.

FIG. 28 depicts the therapeutic effect of VISTA-IgG2a fusion protein onExperimental Autoimmune Encephalomyelitis (EAE) progression. Mice wereimmunized with 175 μg MOG/CFA and pertussis toxin (PT) 300 ng (day 0, 2)to induce active EAE. On day 14, mice were treated with 3 doses per weekof PBS (n=6), 100 μg control IgG2a (n=6), 300 μg control IgG2a (n=6),100 μgVISTA-IgG2a (n=6), or 300 μg mVISTA IgG2a (n=6) (two weeks intotal). The data is shown as the mean±SEM.

FIG. 29 depicts the expression of VISTA healthy human tissues wasexamined by real-time PCR analysis of a cDNA tissue panel (Origene). (A)VISTA was predominantly expressed in haematopoietic tissues or intissues that contain significant numbers of haematopoietic tissues. Thisis consistent with importance of VISTA in immune related functions. (B)The expression pattern of expression was found to follow a similar trendto that of VISTA's closest homologue PD-L1.

FIG. 30 depicts VISTA protein expression in monocytes, dendritic cellsand by approximately 20% of CD4 and CD8 T cells (FIG. 30). VISTAexpression was observed within both of the ‘patrolling’(CD14^(dim)CD16⁺) and ‘inflammatory’ (CD14⁺CD16^(+/−)) subsets of bloodmonocytes, and within both lymphoid and myeloid subsets of dendriticcell.

FIGS. 31A-31D depict the suppression of CFSE dilution of bulk purifiedCD4 (FIG. 31A) and CD8 (FIG. 31B) T cells. An Ig fusion protein wascreated, consisting of the extracellular domain of VISTA and the Fcregion of human IgG containing mutations for reduced Fc receptorbinding. 10 μg/ml of VISTA-Ig or control Ig was immobilized on platesalong with 2.5 μg/ml of anti-CD3 (OKT3) and then proliferation wasmeasured by CFSE dilution.

FIG. 32 depicts the titration of human VISTA-Ig and human VISTA-Ig overdifferent concentrations of OKT3, showed that higher concentrations ofOKT3 can be overcome by higher concentrations of VISTA (FIGS. 32A and32B).

FIG. 33A-D depicts the status of cells was examined following activationin the presence or absence of VISTA-Ig. During 2 days of culture,upregulation by anti-CD3 of the early activation markers CD25 and CD69was blocked by VISTA-Ig (FIGS. 33A & 33B). Similarly, after 5 days ofculture, the shift from expression of CD45RA to CD45RO, indicative ofantigen-experience was prevented (FIG. 33C). VISTA had no affect on cellviability. FIG. 33D shows that VISTA-Ig increased FoxP3 conversion.

FIG. 34 depicts the suppression induced by VISTA where cells werecultured on anti-CD3 and VISTA-Ig for two days, and then moved ontoanti-CD3 alone for 3 days. This further stimulation was unable to rescuesuppression (FIGS. 34A and 34B.)

FIG. 35 shows that VISTA-Ig significantly reduced production of IL-10,TNFα and IFNγ by CD4 (FIG. 35A) and CD8 (FIG. 35B) T cells, and therewas a trend towards a modest decrease in IL-17 production.

FIGS. 36A-36C show that anti-CD28 agonistic antibody provides potentcostimulation to T cells, and so titred into the cultures to challengeVISTA suppression (FIG. 36A-C).

FIG. 37. VISTA mAb treatment reduced tumour growth. Mice were injectedwith A. MB49. B. MCA105. C. EG7 tumour cells, D. ID8-luciferase. E.B16F10. Mice were treated with VISTA mAb 13F3 every other day (300 μg)beginning on day 0 (A-D), or day-2 (E). PD-L1 mAb (MIH5) was alsoadministered to B16F10. Subcutaneous tumour growth was monitored withcalliper and recorded as mm². For intraperitoneal ID8-luciferase tumour,mice were imaged on day 30 and 55 using Xenogen IVIS. For MB49 ELISPOTanalysis (A), tumour drain-LN cells were stimulated with irradiatedtumour cells.

FIG. 38. Human lamina propria DCs express VISTAa. LMPCs isolated fromhealthy colon were stained with biotin-conjugated anti-human VISTA(antibody clone GA1) to identify VISTA expression in Lin-HLA-DR+ LPdendritic cells.

FIGS. 39A-39B Vista-Ig is suppressive to human D4 T cells. CFSE-labeledhuman CD4 T cells were stimulated with plate-bound anti-CD3 at 2.5 μg/mland VISTA-Ig at the indicated concentrations. (A) Representative CFSEdilution profiles. (B) The percentage of CFSE-low cells was quantifiedand shown as mean+/−SEM.

FIGS. 40A-40C VISTA acts in concert with the PD-L1/PD-1 pathway. A.Combinatorial treatment (Day+4) with αVISTA and aPD-L1 mAbs inhibitedB16F10 tumour growth. B. Synergy in vitro: VISTA-Ig and PD-L1-Ig wereimmobilized together with αCD3/CD28 to stimulate CD4+ and CD8+naïve Tcells. Cell proliferation was assessed by CFSE dilution at 72 hrs. C.Differential expression pattern of PD-L1 and VISTA within the TME ofB16F10 tumour. VISTA is expressed only on tumour-infiltrating leukocytes(TILs), whereas PD-L1 is expressed on both tumour cells and TILs.

FIG. 41. Detection of VISTA on myeloid cells with ah VISTA mAb. PBL werestained in the absence (top) or presence of VISTA-Ig (bottom) to confirmspecificity.

FIG. 42 shows that VISTA KO mice exhibit an inflammatory phenotypescharacterized by enhanced inflammatory cytokine level in their serum.

FIG. 43 shows that VISTA KO mice exhibit an inflammatory phenotypescharacterized by the presence of spontaneously activated T cells inblood.

FIG. 44 shows that VISTA KO mice exhibit an inflammatory phenotypescharacterized by enhanced cytokine production of blood T cells.

FIG. 45 shows that VISTA KO mice exhibit an inflammatory phenotypescharacterized by enhanced cytokine production of spleen T cells.

DETAILED DESCRIPTION

We have discovered a novel inhibitory ligand, designated V-domain IgSuppressor of T cell Activation (VISTA), that plays a key role indisrupting protective anti-tumour immunity in mice (1). VISTA bearslimited homology to PDL1, and critically suppresses T cell activationvia an unknown receptor, independent of PD-1. VISTA KO mice displayinflammatory phenotypes, indicating an essential role for VISTA inmaintaining peripheral tolerance. VISTA is highly expressed in thetumour microenvironment (TME) and directly impairs the generation ofoptimal anti-tumour immunity VISTA mAb-mediated blockade significantlysuppressed tumour growth in multiple mouse tumour models.

Based on these findings, we hypothesize that VISTA is expressed ontumour-infiltrating leukocytes, and that this expression is suppressivefor T cell responses in the TME. This application extends our existingstudies of VISTA in mouse models to human patients. Specifically, theapplication further describes examining VISTA expression in patientsamples and testing how this influences T cell function. Based thereon,inhibition of VISTA may be used in the treatment of cancer (similar tothe success with functionally related proteins CTLA-4 and PD-L1). Inanother aspect, the disclosure provides methods of identifying suitableblocking antibodies to be developed for this purpose.

In another aspect, the present invention relates to therapeutic methodsthat modulate the activity and/or which specifically bind or block thebinding of a specific regulatory T cell protein to its counterreceptor.This protein, designated PD-L3 OR VISTA, is a novel andstructurally-distinct, Ig-superfamily inhibitory ligand, whoseextracellular domain bears homology to the B7 family ligand PD-L1. Thismolecule is referred to interchangeably herein as PD-L3 or VISTA or asV-domain Immunoglobulin Suppressor of T cell Activation (VISTA). VISTAis expressed primarily within the hematopoietic compartment and ishighly regulated on myeloid APCs and T cells. Therapeutic interventionof the VISTA inhibitory pathway represents an exciting approach tomodulate T cell-mediated immunity for the treatment of a wide variety ofcancers.

The present invention in particular relates to the use of antibodiesspecific to VISTA or PD-L3 to treat specific cancers includingcolorectal cancer, bladder cancer, ovarian cancer, and melanoma.

As disclosed infra, the expression of VISTA appears to be exclusive tothe hematopoietic compartment and this protein is highly expressed onmature myeloid cells (CD11b^(bright)), with lower levels of expressionon CD4⁺ T cells, T^(reg) and CD8⁺ T cells. Soluble VISTA proteins, e.g.,soluble VISTA-Ig fusion protein, or VISTA expression on APCs, suppressesin vitro CD4⁺ and CD8⁺ T cell proliferation and cytokine production. Itis also observed that anti-VISTA antibodies, e.g., an anti-VISTA mab(13F3) blocked VISTA-induced suppression of T cell responses by VISTA⁺APCs in vitro. Also, it has been discovered that an anti-VISTA mabexacerbated EAE and increased the frequency of encephalitogenic Th17s invivo. Still further, as disclosed in detail infra, it has been foundthat an anti-VISTA mab induces tumor remission in multiple (4) murinetumor models. VISTA expression on myeloid derived suppressor cells(MDSC) in these models is extremely high, suggesting that VISTA MDSCsuppress tumor specific immunity. As shown herein, VISTA exertsimmunosuppressive activities on T cells both in vitro and in vivo, inmouse and in human (in vitro only) and is an important mediator incontrolling the development of autoimmunity and the immune responses tocancer. Specifically, the data show that:

(1) VISTA is a new member of the Ig superfamily and contains an Ig-Vdomain with distant sequence similarity to PD-L1. We disclose hereinthat when produced as an Ig fusion protein or when overexpressed onartificial APCs VISTA inhibits both mouse and human CD4+ and CD8+ T cellproliferation and cytokine production.

(2) VISTA expression on myeloid APCs is inhibitory for T cell responsesin vitro.

(3) VISTA expression on MDSC in the tumor microenvironment is extremelyhigh. Phenotypic and functional analysis of many cell surface moleculespreviously suggested to be involved in MDSC-mediated suppression of Tcells: CD115, CD124, CD80, PD-L1, and PD-L2 were expressed by MDSC butwith no differences in the levels of their expression or proportion ofpositive cells were found between MDSC and cells from tumor-free micethat lack immune suppressive activity. Therefore, we predict that VISTAwill be the primary B7 negative regulator on MDSCs.

(4) Antibody-mediated VISTA blockade induces protective immunity to anautologous tumor.

Based thereon, VISTA appears to be a dominant, negative immuneregulatory molecule on MDSCs that interferes with the development ofprotective anti-tumor immunity. Therefore, blocking the activity of thismolecule with anti-VISTA antibodies will permit the development ofprotective anti-tumor immunity in humans and other mammals.

Therefore, the invention relates to methods of using soluble VISTAproteins, e.g., fusion proteins and multimeric VISTA proteins comprisingmultiple copies of the VISTA extravcelular domain or a fragment thereof,and VISTA binding agents, e.g., small molecules and antibodies orfragments thereof, which bind or modulate (agonize or antagonize) theactivity of VISTA as immune modulators and for the treatment ofdifferent cancers, e.g., colorectal cancer, bladder, ovarian andlymphoma, autoimmune disease, allergy, infection and inflammatoryconditions, e.g. multiple sclerosis and arthritis.

As described in detail infra, VISTA is a novel inhibitory ligand, whichextracellular Ig-V domain bears homology to the two known B7 familyligands Programmed Death Ligand 1 and 2 (PD-L1 and PD-L2) and exhibitsunique sequence features and distinctive expression patterns in vitroand in vivo on subsets of APCs and T cells, (which distinguishes PD-L3or VISTA from other B7 family ligands). This protein has been shown tohave a functional impact on CD4⁺ and CD8⁺ T cell proliferation anddifferentiation (suppresses CD4⁺ and CD8⁺ T cell proliferation, as wellas cytokine production). Based on its expression pattern and inhibitoryimpact on T cells, PD-L3 OR VISTA apparently functions as a regulatoryligand that negatively regulates T cell responses during cognateinteractions between T cells and myeloid derived APCs.

While PD-L3 OR VISTA appears to be a member of the B7 family of ligands,unlike other B7 family ligands, this molecule contains only an Ig-Vdomain without an Ig-C domain, and is phylogenically closer to the B7family receptor Programmed Death-1 (PD-1). Based thereon, PD-L3 ORVISTA, and agonists or antagonists specific thereto can be used toregulate T cell activation and differentiation, and more broadly tomodulate the regulatory network that controls immune responses. Inparticular PD-L3 or VISTA proteins and PD-L3 or VISTA agonists orantagonists, preferably antibodies specific to PD-L3 or VISTA are usefulin modulating immune responses in autoimmunity, inflammatory responsesand diseases, allergy, cancer, infectious disease and transplantation.

Therefore, the present invention in part relates to compositions e.g.,for therapeutic, diagnostic or immune modulatory usage containing anisolated soluble PD-L3 OR VISTA protein or fusion protein, e.g., asoluble VISTA-Ig fusion protein or a multimeric VISTA protein,comprising an amino acid sequence that preferably is at least 70-90%identical to the human or murine PD-L3 OR VISTA polypeptide set forth inSEQ ID NO:2, 4 or 5 or an ortholog, or fragment thereof encoded by agene that specifically hybridizes to SEQ ID NO:1 or 3 that modulatesVISTA in vivo and a pharmaceutically acceptable carrier. In someembodiments, the soluble or multimeric VISTA protein may be directly orindirectly linked to a heterologous (non-VISTA) protein or may beexpressed by a viral vector or a cell containing, e.g., a transfectedimmune cell such as a T cell.

The present invention also provides expression vectors comprising anisolated nucleic acid encoding a VISTA protein that is at least 70-90%identical to the human or murine VISTA amino acid sequence set forth inSEQ ID NO:2, 4 or 5 or a fragment or ortholog thereof, which optionallyis fused to a sequence encoding another protein such as an Igpolypeptide, e.g., an Fc region or a reporter molecule; and host cellscontaining said vectors.

The present invention also specifically relates to an isolated bindingagent, preferably an antibody or antibody fragment which specificallybinds to a PD-L3 OR VISTA protein comprising the amino acid sequence setforth in SEQ ID NO:2, 4 or 5 or a variant, fragment or ortholog thereof.In a preferred embodiment, the binding agent modulates (agonizes orantagonizes) VISTA activity in vitro or in vivo. In most preferredembodiments, the binding agent is an agonistic or antagonistic antibody.

The present invention further provides methods for modulating an immunecell response by contacting an immune cell in vitro or in vivo with aVISTA protein, or binding agent specific thereto, in the presence of aprimary signal so that a response of the immune cell is modulated.(Interaction of VISTA or a modulator thereof transmits a signal toimmune cells, regulating immune responses. PD-L3 OR VISTA protein isexpressed at high levels on myeloid antigen presenting cells, includingmyeloid dendritic cells (DCs) and macrophages, and at lower densities onCD4+ and CD8+ T cells. Upon immune activation, PD-L3 or VISTA expressionis upregulated on myeloid APCs, but downregulated on CD4+ T cells).Therefore, the PD-L3 or VISTA nucleic acids and polypeptides of thepresent invention, and agonists or antagonists thereof are useful, e.g.,in modulating the immune response.

In addition, the PD-L3 or VISTA polypeptides (or biologically activeportions thereof) or modulators of the PD-L3 or VISTA molecules, i.e.,antibodies such as selected using the foregoing methods can beincorporated into pharmaceutical compositions, which optionally includepharmaceutically acceptable carriers.

Immune cells activated in accordance with the method of the instantinvention can subsequently be expanded ex vivo and used in the treatmentand prevention of a variety of diseases; e.g., human T cells which havebeen cloned and expanded in vitro maintain their regulatory activity(Groux, et al. (1997) Nature 389(6652):737-42). Prior to expansion, asource of T cells is obtained from a subject (e.g., a mammals such as ahuman, dog, cat, mouse, rat, or transgenic species thereof). T cells canbe obtained from a number of sources, including peripheral bloodmononuclear cells, bone marrow, lymph node tissue, cord blood, thymustissue, tissue from a site of infection, spleen tissue, tumors or T celllines. T cells can be obtained from a unit of blood collected from asubject using any number of techniques known to the skilled artisan,such as Ficoll™ separation. In another aspect, the present inventionprovides a method for detecting the presence of a PD-L3 or VISTA nucleicacid molecule, protein, or polypeptide in a biological sample bycontacting the biological sample with an agent capable of detecting aPD-L3 OR VISTA nucleic acid molecule, protein, or polypeptide, such thatthe presence of a PD-L3 OR VISTA nucleic acid molecule, protein orpolypeptide is detected in the biological sample. This PD-L3 OR VISTAexpression can be used to detect certain disease sites, includingcancerous sites.

In another aspect, the invention provides a method for modulating PD-L3OR VISTA activity, comprising contacting a cell capable of expressingPD-L3 OR VISTA with an agent that modulates PD-L3 OR VISTA activity,preferably an anti-PD-L3 OR VISTA antibody such that PD-L3 OR VISTAactivity in the cell is modulated. In one embodiment, the agent inhibitsPD-L3 OR VISTA activity. In another embodiment, the agent stimulatesPD-L3 OR VISTA activity. In a further embodiment, the agent interfereswith or enhances the interaction between a PD-L3 OR VISTA polypeptideand its natural binding partner(s). In one embodiment, the agent is anantibody that specifically binds to a PD-L3 OR VISTA polypeptide. Inanother embodiment, the agent is a peptide, peptidomimetic, or othersmall molecule that binds to a PD-L3 OR VISTA polypeptide.

In still another embodiment, the agent modulates expression of PD-L3 ORVISTA by modulating transcription of a PD-L3 OR VISTA gene, translationof a PD-L3 OR VISTA mRNA, or post-translational modification of a PD-L3OR VISTA polypeptide. In another embodiment, the agent is a nucleic acidmolecule having a nucleotide sequence that is antisense to the codingstrand of a PD-L3 OR VISTA mRNA or a PD-L3 OR VISTA gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder or condition characterized byaberrant, insufficient, or unwanted PD-L3 OR VISTA polypeptide ornucleic acid expression or activity by administering an agent which is aPD-L3 OR VISTA modulator to the subject. In one preferred embodiment,the PD-L3 OR VISTA modulator is a PD-L3 OR VISTA polypeptide, preferablya soluble fusion protein or multimeric VISTA protein or anti-VISTAantibody as described infra. In another embodiment the PD-L3 OR VISTAmodulator is a PD-L3 OR VISTA nucleic acid molecule, e,g in anadenoviral vector. In another embodiment, the invention further providestreating the subject with an additional agent that modulates an immuneresponse.

In still another embodiment, the invention provides a vaccine comprisingan antigen and an agent that modulates (enhances or inhibits) PD-L3 ORVISTA activity. In a preferred embodiment, the vaccine inhibits theinteraction between PD-L3 OR VISTA and its natural binding partner(s).

In another aspect the invention provides methods for identifying acompound that binds to or modulates the activity of a PD-L3 OR VISTApolypeptide, by providing an indicator composition comprising a PD-L3 ORVISTA polypeptide having PD-L3 OR VISTA activity, contacting theindicator composition with a test compound, and determining the effectof the test compound on PD-L3 OR VISTA activity in the indicatorcomposition to identify a compound that modulates the activity of aPD-L3 OR VISTA polypeptide.

In one aspect, the invention features a method for modulating theinteraction of PD-L3 OR VISTA with its natural binding partner(s) on animmune cell comprising contacting an antigen presenting cell whichexpresses PD-L3 OR VISTA with an agent selected from the groupconsisting of: a form of PD-L3 OR VISTA, or an agent that modulates theinteraction of PD-L3 OR VISTA and its natural binding partner(s) suchthat the interaction of PD-L3 OR VISTA with it natural bindingpartner(s) on an immune cell is modulated. In a preferred embodiment, anagent that modulates the interaction of PD-L3 OR VISTA and its naturalbinding partner(s) is an antibody that specifically binds to PD-L3 ORVISTA. In one embodiment, the interaction of PD-L3 OR VISTA with itsnatural binding partner(s) is upregulated. In another embodiment, theinteraction of PD-L3 OR VISTA with its natural binding partner(s) isdownregulated. In one embodiment, the method further comprisescontacting the immune cell or the antigen presenting cell with anadditional agent that modulates an immune response.

In one embodiment, the step of contacting is performed in vitro. Inanother embodiment, the step of contacting is performed in vivo. In oneembodiment, the immune cell is selected from the group consisting of: aT cell, a monocyte, a macrophage, a dendritic cell, a B cell, and amyeloid cell.

In another aspect, the invention pertains to a method for inhibiting orincreasing activation in an immune cell comprising increasing orinhibiting the activity or expression of PD-L3 OR VISTA in a cell suchthat immune cell activation is inhibited or increased.

In yet another aspect, the invention pertains to a vaccine comprising anantigen and an agent that inhibits the interaction between PD-L3 ORVISTA and its natural binding partner(s).

In another aspect, the invention pertains to a method for treating asubject having a condition that would benefit from upregulation of animmune response comprising administering an agent that inhibits theinteraction between PD-L3 OR VISTA and its natural binding partner(s) onimmune cells of the subject such that a condition that would benefitfrom upregulation of an immune response is treated. In one preferredembodiment, the agent comprises a blocking antibody or a small moleculethat binds to PD-L3 OR VISTA and inhibits the interaction between PD-L3OR VISTA and its natural binding partner(s). In another embodiment, themethod further comprises administering a second agent that upregulatesan immune response to the subject. In another aspect, the inventionpertains to a method for treating a subject having a condition thatwould benefit from downregulation of an immune response comprisingadministering an agent that stimulates the interaction between PD-L3 ORVISTA and its natural binding partner(s) on cells of the subject suchthat a condition that would benefit from downregulation of an immuneresponse is treated.

For example the condition treated with the PD-L3 OR VISTA protein orbinding agents is selected from the group consisting of: a tumor, apathogenic infection, an inflammatory immune response or condition,preferably less pronounced inflammatory conditions, or animmunosuppressive disease. Specific examples include multiple sclerosis,thyroiditis, rheumatoid arthritis, diabetes type II and type I andcancers, both advanced and early forms, including metastatic cancerssuch as colorectal cancer, bladder cancer, ovarian cancer, melanoma,lung cancer, and other cancers wherein VISTA suppresses an effectiveanti-tumor response. In some case the individual may be administeredcells or a viral vector that express a nucleic acid that encodes ananti-VISTA antibody or VISTA fusion protein.

Exemplary conditions treatable using PD-L3 OR VISTA proteins, bindingagents or PD-L3 OR VISTA antagonists or agonists according to theinvention include by way of example transplant, an allergy, infectiousdisease, cancer, and inflammatory or autoimmune disorders, e.g., aninflammatory immune disorder. Specific examples of the foregoing includetype 1 diabetes, multiple sclerosis, rheumatoid arthritis, psoriaticarthritis, systemic lupus erythematosis, rheumatic diseases, allergicdisorders, asthma, allergic rhinitis, skin disorders, gastrointestinaldisorders such as Crohn's disease and ulcerative colitis, transplantrejection, poststreptococcal and autoimmune renal failure, septic shock,systemic inflammatory response syndrome (SIRS), adult respiratorydistress syndrome (ARDS) and envenomation; autoinflammatory diseases aswell as degenerative bone and joint diseases including osteoarthritis,crystal arthritis and capsulitis and other arthropathies. Further, themethods and compositions can be used for treating tendonitis,ligamentitis and traumatic joint injury.

In preferred embodiments the subject PD-L3 OR VISTA proteins, nucleiacids, and ligands specific to PD-L3 OR VISTA, preferably antibodieshaving desired effects on PD-L3 OR VISTA functions are used to treatconditions such a cancer, autoimmune diseases, allergy, inflammatorydisorders or infection and more specifically immune system disorderssuch as severe combined immunodeficiency, multiple sclerosis, systemiclupus erythematosus, type I diabetes mellitus, lymphoproliferativesyndrome, inflammatory bowel disease, allergies, asthma,graft-versus-host disease, and transplant rejection; immune responses toinfectious pathogens such as bacteria and viruses; and immune systemcancers such as lymphomas and leukemias)

In addition to the infectious and parasitic agents mentioned above,another area for desirable enhanced immunogenicity to a non-infectiousagent is in the area of dysproliferative diseases, including but notlimited to cancer, in which cells expressing cancer antigens aredesirably eliminated from the body. Tumor antigens which can be used inthe compositions and methods of the invention include, but are notlimited to, prostate specific antigen (PSA), breast cancer antigens,bladder cancer antigens, ovarian cancer antigens, testicular cancerantigens, melanoma antigens, colorectal cancer antigens, telomerase;multidrug resistance proteins such as P-glycoprotein; MAGE-1, alphafetoprotein, carcinoembryonic antigen, mutant p53, papillomavirusantigens, gangliosides or other carbohydrate-containing components ofmelanoma or other tumor cells. It is contemplated by the invention thatantigens from any type of tumor cell can be used in the compositions andmethods described herein. The antigen may be a cancer cell, orimmunogenic materials isolated from a cancer cell, such as membraneproteins. Included are survivin and telomerase universal antigens andthe MAGE family of cancer testis antigens. Antigens which have beenshown to be involved in autoimmunity and could be used in the methods ofthe present invention to induce tolerance include, but are not limitedto, myelin basic protein, myelin oligodendrocyte glycoprotein andproteolipid protein of multiple sclerosis and CII collagen protein ofrheumatoid arthritis.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein may be used inthe invention or testing of the present invention, suitable methods andmaterials are described herein. The materials, methods and examples areillustrative only, and are not intended to be limiting.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer, lungcancer (including small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung), cancerof the peritoneum, hepatocellular cancer, gastric or stomach cancer(including gastrointestinal cancer), pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; multiple myeloma and post-transplant lymphoproliferativedisorder (PTLD).

Exemplary cancers amenable for treatment by the present inventioninclude, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,and leukemia or lymphoid malignancies. More particular examples of suchcancers include colorectal, bladder, ovarian, melanoma, squamous cellcancer, lung cancer (including small-cell lung cancer, non-small celllung cancer, adenocarcinoma of the lung, and squamous carcinoma of thelung), cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer (including gastrointestinal cancer), pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidney orrenal cancer, liver cancer, prostate cancer, vulval cancer, thyroidcancer, hepatic carcinoma and various types of head and neck cancer, aswell as B-cell lymphoma (including low grade/follicular non-Hodgkin'slymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; and post-transplantlymphoproliferative disorder (PTLD), as well as abnormal vascularproliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), and Meigs' syndrome. Preferably, thecancer is selected from the group consisting of colorectal cancer,breast cancer, colorectal cancer, rectal cancer, non-small cell lungcancer, non-Hodgkins lymphoma (NHL), renal cell cancer, prostate cancer,liver cancer, pancreatic cancer, soft-tissue sarcoma, kaposi's sarcoma,carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer,mesothelioma, and multiple myeloma. In an exemplary embodiment thecancer is an early or advanced (including metastatic) bladder, ovarianor melanoma. In another embodiment the cancer is colorectal cancer. Thecancerous conditions amenable for treatment of the invention includemetastatic cancers wherein VISTA expression by myeloid derivedsuppressor cells suppress antitumor responses and anti-invasive immuneresponses. The method of the present invention is particularly suitablefor the treatment of vascularized tumors.

The invention is also suitable for treating cancers in combination withchemotherapy or radiotherapy or other biologics and for enhancing theactivity thereof, i.e., in individuals wherein VISTA expression bymyeloid derived suppressor cells suppress antitumor responses and theefficacy of chemotherapy or radiotherapy or biologic efficacy. Anychemotherapeutic agent exhibiting anticancer activity can be usedaccording to the present invention. Preferably, the chemotherapeuticagent is selected from the group consisting of alkylating agents,antimetabolites, folic acid analogs, pyrimidine analogs, purine analogsand related inhibitors, vinca alkaloids, epipodopyyllotoxins,antibiotics, L-Asparaginase, topoisomerase inhibitor, interferons,platinum coordination complexes, anthracenedione substituted urea,methyl hydrazine derivatives, adrenocortical suppressant,adrenocorticosteroides, progestins, estrogens, antiestrogen, androgens,antiandrogen, and gonadotropin-releasing hormone analog. Morepreferably, the chemotherapeutic agent is selected from the groupconsisting of 5-fluorouracil (5-FU), leucovorin (LV), irenotecan,oxaliplatin, capecitabine, paclitaxel and doxetaxel. Two or morechemotherapeutic agents can be used in a cocktail to be administered incombination with administration of the anti-VEGF antibody. One preferredcombination chemotherapy is fluorouracil-based, comprising 5-FU and oneor more other chemotherapeutic agent(s). Suitable dosing regimens ofcombination chemotherapies are known in the art and described in, forexample, Saltz et al. (1999) Proc ASCO 18:233a and Douillard et al.(2000) Lancet 355:1041-7. The bilogic may be another immune potentiatorssuch as antibodies to PD-L1, PD-L2, CTLA-4 and PD-L1, PD-L2, CTLA-4fusion proteins as well as cytokines, growth factor antagonists andagonists, hormones and anti-cytokine antibodies.

“Activating receptor,” as used herein, refers broadly to immune cellreceptors that bind antigen, complexed antigen (e.g., in the context ofMHC molecules), Ig-fusion proteins, ligands, or antibodies. Activatingreceptors but are not limited to T cell receptors (TCRs), B cellreceptors (BCRs), cytokine receptors, LPS receptors, complementreceptors, and Fc receptors. For example, T cell receptors are presenton T cells and are associated with CD3 molecules. T cell receptors arestimulated by antigen in the context of MHC molecules (as well as bypolyclonal T cell activating reagents). T cell activation via the TCRresults in numerous changes, e.g., protein phosphorylation, membranelipid changes, ion fluxes, cyclic nucleotide alterations, RNAtranscription changes, protein synthesis changes, and cell volumechanges. For example, T cell receptors are present on T cells and areassociated with CD3 molecules. T cell receptors are stimulated byantigen in the context of MHC molecules (as well as by polyclonal T cellactivating reagents). T cell activation via the TCR results in numerouschanges, e.g, protein phosphorylation, membrane lipid changes, ionfluxes, cyclic nucleotide alterations, RNA transcription changes,protein synthesis changes, and cell volume changes.

“Antigen presenting cell,” as used herein, refers broadly toprofessional antigen presenting cells (e.g., B lymphocytes, monocytes,dendritic cells, and Langerhans cells) as well as other antigenpresenting cells (e.g., keratinocytes, endothelial cells, astrocytes,fibroblasts, and oligodendrocytes).

“Amino acid,” as used herein refers broadly to naturally occurring andsynthetic amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally occurring amino acids are those encoded by thegenetic code, as well as those amino acids that are later modified(e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine.) Aminoacid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid (i.e., an a carbon that isbound to a hydrogen, a carboxyl group, an amino group), and an R group(e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium.) Analogs may have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

“Anergy” or “tolerance,” as used herein, refers broadly to refractivityto activating receptor-mediated stimulation. Refractivity is generallyantigen-specific and persists after exposure to the tolerizing antigenhas ceased. For example, anergy in T cells (as opposed tounresponsiveness) is characterized by lack of cytokine production, e.g.,IL-2. T cell anergy occurs when T cells are exposed to antigen andreceive a first signal (a T cell receptor or CD-3 mediated signal) inthe absence of a second signal (a costimulatory signal). Under theseconditions, reexposure of the cells to the same antigen (even ifreexposure occurs in the presence of a costimulatory molecule) resultsin failure to produce cytokines and, thus, failure to proliferate.Anergic T cells can, however, mount responses to unrelated antigens andcan proliferate if cultured with cytokines (e.g., IL-2). For example, Tcell anergy can also be observed by the lack of IL-2 production by Tlymphocytes as measured by ELISA or by a proliferation assay using anindicator cell line. Alternatively, a reporter gene construct can beused. For example, anergic T cells fail to initiate IL-2 genetranscription induced by a heterologous promoter under the control ofthe 5′ IL-2 gene enhancer or by a multimer of the AP1 sequence that canbe found within the enhancer (Kang et al. (1992) Science 257:1134).Modulation of a costimulatory signal results in modulation of effectorfunction of an immune cell. Thus, the term “PD-L3 OR VISTA activity”includes the ability of a PD-L3 OR VISTA polypeptide to bind its naturalbinding partner(s), the ability to modulate immune cell costimulatory orinhibitory signals, and the ability to modulate the immune response.Modulation of an inhibitory signal in an immune cell results inmodulation of proliferation of and/or cytokine secretion by an immunecell.

“Antibody”, as used herein, refers broadly to an “antigen-bindingportion” of an antibody (also used interchangeably with “antibodyportion,” “antigen-binding fragment,” “antibody fragment”), as well aswhole antibody molecules. The term “antigen-binding portion”, as usedherein, refers to one or more fragments of an antibody that retain theability to specifically bind to an antigen (e.g, VISTA (PD-L3)). Theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Examples of antigen-binding fragmentsencompassed within the term “antigen-binding portion” of an antibodyinclude (a) a Fab fragment, a monovalent fragment consisting of the VL,VH, CL and CH1 domains; (b) a F(ab′)2 fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (c) a Fd fragment consisting of the VH and CH1 domains; (d) a Fvfragment consisting of the VL and VH domains of a single arm of anantibody; (e) a dAb fragment (Ward, et al. (1989) Nature 341: 544-546),which consists of a VH domain; and (f) an isolated complementarilydetermining region (CDR). Furthermore, although the two domains of theFv fragment, VL and VH, are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules (known as single chain Fv (scFv). Seee.g., Bird, et al. (1988) Science 242: 423-426; Huston, et al. (1988)Proc Natl. Acad. Sci. USA 85: 5879-5883; and Osbourn, et al. (1998) Nat.Biotechnol. 16: 778. Single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.Any VH and VL sequences of specific scFv can be linked to humanimmunoglobulin constant region cDNA or genomic sequences, in order togenerate expression vectors encoding complete IgG molecules or otherisotypes. VH and Vl can also be used in the generation of Fab, Fv, orother fragments of immunoglobulins using either protein chemistry orrecombinant DNA technology. Other forms of single chain antibodies, suchas diabodies are also encompassed. Diabodies are bivalent, bispecificantibodies in which VH and VL domains are expressed on a singlepolypeptide chain, but using a linker that is too short to allow forpairing between the two domains on the same chain, thereby forcing thedomains to pair with complementary domains of another chain and creatingtwo antigen binding sites. See e.g., Holliger, et al. (1993) Proc Natl.Acad. Sci. USA 90: 6444-6448; Poljak, et al. (1994) Structure 2:1121-1123.

Still further, an antibody or antigen-binding portion thereof(antigen-binding fragment, antibody fragment, antibody portion) may bepart of a larger immunoadhesion molecules, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of immunoadhesion moleculesinclude use of the streptavidin core region to make a tetrameric scFvmolecule (Kipriyanov, et al. (1995) Hum. Antibodies Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminalpolyhistidine tag to make bivalent and biotinylated scFv molecules.Kipriyanov, et al. (1994) Mol Immunol. 31: 1047-1058. Antibody portions,such as Fab and F(ab′)2 fragments, can be prepared from whole antibodiesusing conventional techniques, such as papain or pepsin digestion,respectively, of whole antibodies. Moreover, antibodies, antibodyportions and immunoadhesion molecules can be obtained using standardrecombinant DNA techniques, as described herein.

Antibodies may be polyclonal, monoclonal, xenogeneic, allogeneic,syngeneic, or modified forms thereof, e.g., humanized, chimeric.Preferably, antibodies of the invention bind specifically orsubstantially specifically to VISTA (PD-L3) molecules. The terms“monoclonal antibodies” and “monoclonal antibody composition”, as usedherein, refer to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope of an antigen, whereas the term “polyclonalantibodies” and “polyclonal antibody composition” refer to a populationof antibody molecules that contain multiple species of antigen bindingsites capable of interacting with a particular antigen. A monoclonalantibody composition, typically displays a single binding affinity for aparticular antigen with which it immunoreacts.

“Antigen,” as used herein, refers broadly to a molecule or a portion ofa molecule capable of being bound by an antibody which is additionallycapable of inducing an animal to produce an antibody capable of bindingto an epitope of that antigen. An antigen may have one epitope, or havemore than one epitope. The specific reaction referred to hereinindicates that the antigen will react, in a highly selective manner,with its corresponding antibody and not with the multitude of otherantibodies which may be evoked by other antigens. In the case of adesired enhanced immune response to particular antigens of interest,antigens include, but are not limited to, infectious disease antigensfor which a protective immune response may be elicited are exemplary.

“Allergic disease,” as used herein, refers broadly to a diseaseinvolving allergic reactions. More specifically, an “allergic disease”is defined as a disease for which an allergen is identified, where thereis a strong correlation between exposure to that allergen and the onsetof pathological change, and where that pathological change has beenproven to have an immunological mechanism. Herein, an immunologicalmechanism means that leukocytes show an immune response to allergenstimulation.

“Antisense nucleic acid molecule,” as used herein, refers broadly to anucleotide sequence which is complementary to a “sense” nucleic acidencoding a protein (e.g., complementary to the coding strand of adouble-stranded cDNA molecule) complementary to an mRNA sequence orcomplementary to the coding strand of a gene. Accordingly, an antisensenucleic acid molecule can hydrogen bond to a sense nucleic acidmolecule.

“Asthma,” as used herein, refers broadly to a disorder of therespiratory system characterized by inflammation, narrowing of theairways and increased reactivity of the airways to inhaled agents.Asthma is frequently, although not exclusively, associated with atopicor allergic symptoms.

“Apoptosis,” as used herein, refers broadly to programmed cell deathwhich can be characterized using techniques which are known in the art.Apoptotic cell death can be characterized by cell shrinkage, membraneblebbing, and chromatin condensation culminating in cell fragmentation.Cells undergoing apoptosis also display a characteristic pattern ofinternucleosomal DNA cleavage.

“Autoimmunity” or “autoimmune disease or condition,” as used herein,refers broadly to a disease or disorder arising from and directedagainst an individual's own tissues or a co-segregate or manifestationthereof or resulting condition therefrom.

“B cell receptor” (BCR),” as used herein, refers broadly to the complexbetween membrane Ig (mIg) and other transmembrane polypeptides (e.g.,Igα and Igβ) found on B cells. The signal transduction function of mIgis triggered by crosslinking of receptor molecules by oligomeric ormultimeric antigens. B cells can also be activated byanti-immunoglobulin antibodies. Upon BCR activation, numerous changesoccur in B cells, including tyrosine phosphorylation.

“Cancer,” as used herein, refers broadly to any neoplastic disease(whether invasive or metastatic) characterized by abnormal anduncontrolled cell division causing malignant growth or tumor (e.g.,unregulated cell growth.)

“Chimeric antibody,” as used herein, refers broadly to an antibodymolecule in which the constant region, or a portion thereof, is altered,replaced or exchanged so that the antigen binding site (variable region)is linked to a constant region of a different or altered class, effectorfunction and/or species, or an entirely different molecule which confersnew properties to the chimeric antibody, e.g., an enzyme, toxin,hormone, growth factor, drug, the variable region or a portion thereof,is altered, replaced or exchanged with a variable region having adifferent or altered antigen specificity.

“Coding region,” as used herein, refers broadly to regions of anucleotide sequence comprising codons which are translated into aminoacid residues, whereas the term “noncoding region” refers to regions ofa nucleotide sequence that are not translated into amino acids (e.g., 5′and 3′ untranslated regions).

“Conservatively modified variants,” as used herein, applies to bothamino acid and nucleic acid sequences, and with respect to particularnucleic acid sequences, refers broadly to conservatively modifiedvariants refers to those nucleic acids which encode identical oressentially identical amino acid sequences, or where the nucleic aciddoes not encode an amino acid sequence, to essentially identicalsequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode any given protein.“Silent variations” are one species of conservatively modified nucleicacid variations. Every nucleic acid sequence herein which encodes apolypeptide also describes every possible silent variation of thenucleic acid. One of skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine, andTGG, which is ordinarily the only codon for tryptophan) may be modifiedto yield a functionally identical molecule.

“Complementarity determining region,” “hypervariable region,” or “CDR,”as used herein, refers broadly to one or more of the hyper-variable orcomplementarily determining regions (CDRs) found in the variable regionsof light or heavy chains of an antibody. See Kabat, et al. (1987)“Sequences of Proteins of Immunological Interest” National Institutes ofHealth, Bethesda, Md. These expressions include the hypervariableregions as defined by Kabat, et al. (1983) “Sequences of Proteins ofImmunological Interest” U.S. Dept. of Health and Human Services or thehypervariable loops in 3-dimensional structures of antibodies. Chothiaand Lesk (1987) J Mol. Biol. 196: 901-917. The CDRs in each chain areheld in close proximity by framework regions and, with the CDRs from theother chain, contribute to the formation of the antigen binding site.Within the CDRs there are select amino acids that have been described asthe selectivity determining regions (SDRs) which represent the criticalcontact residues used by the CDR in the antibody-antigen interaction.Kashmiri (2005) Methods 36: 25-34.

“Control amount,” as used herein, refers broadly to a marker can be anyamount or a range of amounts to be compared against a test amount of amarker. For example, a control amount of a marker may be the amount of amarker in a patient with a particular disease or condition or a personwithout such a disease or condition. A control amount can be either inabsolute amount (e.g., microgram/10 or a relative amount (e.g., relativeintensity of signals).

“Costimulatory receptor,” as used herein, refers broadly to receptorswhich transmit a costimulatory signal to an immune cell, e.g., CD28 orICOS. As used herein, the term “inhibitory receptors” includes receptorswhich transmit a negative signal to an immune cell.

“Costimulate,” as used herein, refers broadly to the ability of acostimulatory molecule to provide a second, non-activating,receptor-mediated signal (a “costimulatory signal”) that inducesproliferation or effector function. For example, a costimulatory signalcan result in cytokine secretion (e.g., in a T cell that has received aT cell-receptor-mediated signal.) Immune cells that have received a cellreceptor-mediated signal (e.g., via an activating receptor) may bereferred to herein as “activated immune cells.”

“Cytoplasmic domain,” as used herein, refers broadly to the portion of aprotein which extends into the cytoplasm of a cell.

“Diagnostic,” as used herein, refers broadly to identifying the presenceor nature of a pathologic condition. Diagnostic methods differ in theirsensitivity and specificity. The “sensitivity” of a diagnostic assay isthe percentage of diseased individuals who test positive (percent of“true positives”). Diseased individuals not detected by the assay are“false negatives.” Subjects who are not diseased and who test negativein the assay are termed “true negatives.” The “specificity” of adiagnostic assay is 1 minus the false positive rate, where the “falsepositive” rate is defined as the proportion of those without the diseasewho test positive. While a particular diagnostic method may not providea definitive diagnosis of a condition, it suffices if the methodprovides a positive indication that aids in diagnosis.

“Diagnosing,” as used herein refers broadly to classifying a disease ora symptom, determining a severity of the disease, monitoring diseaseprogression, forecasting an outcome of a disease and/or prospects ofrecovery. The term “detecting” may also optionally encompass any of theforegoing. Diagnosis of a disease according to the present inventionmay, in some embodiments, be affected by determining a level of apolynucleotide or a polypeptide of the present invention in a biologicalsample obtained from the subject, wherein the level determined can becorrelated with predisposition to, or presence or absence of thedisease. It should be noted that a “biological sample obtained from thesubject” may also optionally comprise a sample that has not beenphysically removed from the subject.

“Effective amount,” as used herein, refers broadly to the amount of acompound, antibody, antigen, or cells that, when administered to apatient for treating a disease, is sufficient to effect such treatmentfor the disease. The effective amount may be an amount effective forprophylaxis, and/or an amount effective for prevention. The effectiveamount may be an amount effective to reduce, an amount effective toprevent the incidence of signs/symptoms, to reduce the severity of theincidence of signs/symptoms, to eliminate the incidence ofsigns/symptoms, to slow the development of the incidence ofsigns/symptoms, to prevent the development of the incidence ofsigns/symptoms, and/or effect prophylaxis of the incidence ofsigns/symptoms. The “effective amount” may vary depending on the diseaseand its severity and the age, weight, medical history, susceptibility,and pre-existing conditions, of the patient to be treated. The term“effective amount” is synonymous with “therapeutically effective amount”for purposes of this invention.

“Extracellular domain,” as used herein refers broadly to the portion ofa protein that extend from the surface of a cell.

“Expression vector,” as used herein, refers broadly to any recombinantexpression system for the purpose of expressing a nucleic acid sequenceof the invention in vitro or in vivo, constitutively or inducibly, inany cell, including prokaryotic, yeast, fungal, plant, insect ormammalian cell. The term includes linear or circular expression systems.The term includes expression systems that remain episomal or integrateinto the host cell genome. The expression systems can have the abilityto self-replicate or not, i.e., drive only transient expression in acell. The term includes recombinant expression cassettes which containonly the minimum elements needed for transcription of the recombinantnucleic acid.

“Family,” as used herein, refers broadly to the polypeptide and nucleicacid molecules of the invention is intended to mean two or morepolypeptide or nucleic acid molecules having a common structural domainor motif and having sufficient amino acid or nucleotide sequencehomology as defined herein. Family members can be naturally ornon-naturally occurring and can be from either the same or differentspecies. For example, a family can contain a first polypeptide of humanorigin, as well as other, distinct polypeptides of human origin oralternatively, can contain homologues of non-human origin (e.g., monkeypolypeptides.) Members of a family may also have common functionalcharacteristics.

“Fc receptor” (FcRs) as used herein, refers broadly to cell surfacereceptors for the Fc portion of immunoglobulin molecules (Igs). Fcreceptors are found on many cells which participate in immune responses.Among the human FcRs that have been identified so far are those whichrecognize IgG (designated FcγR), IgE (FcεR1), IgA (FeαR), andpolymerized IgM/A (FcμαR). FcRs are found in the following cell types:FcεRI (mast cells), FcεRII (many leukocytes), FcαR (neutrophils), andFcμαR (glandular epithelium, hepatocytes). Hogg (1988) Immunol Today 9:185-86. The widely studied FcγRs are central in cellular immunedefenses, and are responsible for stimulating the release of mediatorsof inflammation and hydrolytic enzymes involved in the pathogenesis ofautoimmune disease. Unkeless (1988) Annu. Rev. Immunol. 6: 251-87. TheFcγRs provide a crucial link between effector cells and the lymphocytesthat secrete Ig, since the macrophage/monocyte, polymorphonuclearleukocyte, and natural killer (NK) cell Fc gamma Rs confer an element ofspecific recognition mediated by IgG. Human leukocytes have at leastthree different receptors for IgG: hFcγRI (found onmonocytes/macrophages), hFcγRII (on monocytes, neutrophils, eosinophils,platelets, possibly B cells, and the K562 cell line), and FcγIII (on NKcells, neutrophils, eosinophils, and macrophages).

With respect to T cells, transmission of a costimulatory signal to a Tcell involves a signaling pathway that is not inhibited by cyclosporinA. In addition, a costimulatory signal can induce cytokine secretion(e.g., IL-2 and/or IL-10) in a T cell and/or can prevent the inductionof unresponsiveness to antigen, the induction of anergy, or theinduction of cell death in the T cell.

“Framework region” or “FR,” as used herein, refers broadly to one ormore of the framework regions within the variable regions of the lightand heavy chains of an antibody. See Kabat, et al. (1987) “Sequences ofProteins of Immunological Interest” National Institutes of Health,Bethesda, Md. These expressions include those amino acid sequenceregions interposed between the CDRs within the variable regions of thelight and heavy chains of an antibody.

“Heterologous,” as used herein, refers broadly to portions of a nucleicacid indicates that the nucleic acid comprises two or more subsequencesthat are not found in the same relationship to each other in nature. Forinstance, the nucleic acid is typically recombinantly produced, havingtwo or more sequences from unrelated genes arranged to make a newfunctional nucleic acid (e.g., a promoter from one source and a codingregion from another source.) Similarly, a heterologous protein indicatesthat the protein comprises two or more subsequences that are not foundin the same relationship to each other in nature (e.g., a fusionprotein).

“High affinity,” as used herein, refers broadly to an antibody having aKD of at least 10⁻⁸ M, more preferably at least 10⁻⁹ M and even morepreferably at least 10⁻¹⁰ M for a target antigen. However, “highaffinity” binding can vary for other antibody isotypes. For example,“high affinity” binding for an IgM isotype refers to an antibody havinga KD of at least 10⁻⁷ M, more preferably at least 10⁻⁸ M.

“Homology,” as used herein, refers broadly to a degree of similaritybetween a nucleic acid sequence and a reference nucleic acid sequence orbetween a polypeptide sequence and a reference polypeptide sequence.Homology may be partial or complete. Complete homology indicates thatthe nucleic acid or amino acid sequences are identical. A partiallyhomologous nucleic acid or amino acid sequence is one that is notidentical to the reference nucleic acid or amino acid sequence. Thedegree of homology can be determined by sequence comparison. The term“sequence identity” may be used interchangeably with “homology.”

“Host cell,” as used herein, refers broadly to refer to a cell intowhich a nucleic acid molecule of the invention, such as a recombinantexpression vector of the invention, has been introduced. Host cells maybe prokaryotic cells (e.g., E. coli), or eukaryotic cells such as yeast,insect (e.g., SF9), amphibian, or mammalian cells such as CHO, HeLa,HEK-293, e.g., cultured cells, explants, and cells in vivo. The terms“host cell” and “recombinant host cell” are used interchangeably herein.It should be understood that such terms refer not only to the particularsubject cell but to the progeny or potential progeny of such a cell.Because certain modifications may occur in succeeding generations due toeither mutation or environmental influences, progeny may not, in fact,be identical to the parent cell, but are still included within the scopeof the term as used herein.

“Humanized antibody,” as used herein, refers broadly to includeantibodies made by a non-human cell having variable and constant regionswhich have been altered to more closely resemble antibodies that wouldbe made by a human cell. For example, by altering the non-human antibodyamino acid sequence to incorporate amino acids found in human germlineimmunoglobulin sequences. The humanized antibodies of the invention mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo), for example in theCDRs. The term “humanized antibody”, as used herein, also includesantibodies in which CDR sequences derived from the germline of anothermammalian species, such as a mouse, have been grafted onto humanframework sequences.

“Hybridization,” as used herein, refers broadly to the physicalinteraction of complementary (including partially complementary)polynucleotide strands by the formation of hydrogen bonds betweencomplementary nucleotides when the strands are arranged antiparallel toeach other.

“IgV domain” and “IgC domain” as used herein, refer broadly to Igsuperfamily member domains. These domains correspond to structural unitsthat have distinct folding patterns called Ig folds. Ig folds arecomprised of a sandwich of two beta sheets, each consisting ofantiparallel beta strands of 5-10 amino acids with a conserved disulfidebond between the two sheets in most, but not all, domains. IgC domainsof Ig, TCR, and MHC molecules share the same types of sequence patternsand are called the C1 set within the Ig superfamily. Other IgC domainsfall within other sets. IgV domains also share sequence patterns and arecalled V set domains. IgV domains are longer than C-domains and form anadditional pair of beta strands.

“Immune cell,” as used herein, refers broadly to cells that are ofhematopoietic origin and that play a role in the immune response. Immunecells include lymphocytes, such as B cells and T cells; natural killercells; and myeloid cells, such as monocytes, macrophages, eosinophils,mast cells, basophils, and granulocytes.

“Immunoassay,” as used herein, refers broadly to an assay that uses anantibody to specifically bind an antigen. The immunoassay may becharacterized by the use of specific binding properties of a particularantibody to isolate, target, and/or quantify the antigen.

“Immune response,” as used herein, refers broadly to T cell-mediatedand/or B cell-mediated immune responses that are influenced bymodulation of T cell costimulation. Exemplary immune responses include Bcell responses (e.g., antibody production) T cell responses (e.g.,cytokine production, and cellular cytotoxicity) and activation ofcytokine responsive cells, e.g., macrophages. As used herein, the term“downmodulation” with reference to the immune response includes adiminution in any one or more immune responses, while the term“upmodulation” with reference to the immune response includes anincrease in any one or more immune responses. It will be understood thatupmodulation of one type of immune response may lead to a correspondingdownmodulation in another type of immune response. For example,upmodulation of the production of certain cytokines (e.g., IL-10) canlead to downmodulation of cellular immune responses.

“Inflammatory conditions or inflammatory disease,” as used herein,refers broadly to chronic or acute inflammatory diseases.

“Inhibitory signal,” as used herein, refers broadly to a signaltransmitted via an inhibitory receptor molecule on an immune cell. Asignal antagonizes a signal via an activating receptor (e.g., via a TCR,CD3, BCR, or Fc molecule) and can result, e.g., in inhibition of: secondmessenger generation; proliferation; or effector function in the immunecell, e.g., reduced phagocytosis, antibody production, or cellularcytotoxicity, or the failure of the immune cell to produce mediators(e.g., cytokines (e.g., IL-2) and/or mediators of allergic responses);or the development of anergy.

“Isolated,” as used herein, refers broadly to material removed from itsoriginal environment in which it naturally occurs, and thus is alteredby the hand of man from its natural environment. Isolated material maybe, for example, exogenous nucleic acid included in a vector system,exogenous nucleic acid contained within a host cell, or any materialwhich has been removed from its original environment and thus altered bythe hand of man (e.g., “isolated antibody”). For example, “isolated” or“purified,” as used herein, refers broadly to a protein, DNA, antibody,RNA, or biologically active portion thereof, that is substantially freeof cellular material or other contaminating proteins from the cell ortissue source from which the biological substance is derived, orsubstantially free from chemical precursors or other chemicals whenchemically synthesized. The language “substantially free of cellularmaterial” includes preparations of VISTA (PD-L3) protein in which theprotein is separated from cellular components of the cells from which itis isolated or recombinantly produced.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds PD-L3 OR VISTA is substantially free of antibodies thatspecifically bind antigens other than PD-L3 OR VISTA). Moreover, anisolated antibody may be substantially free of other cellular materialand/or chemicals.

“K-assoc” or “Ka”, as used herein, refers broadly to the associationrate of a particular antibody-antigen interaction, whereas the term“Kdiss” or “Kd,” as used herein, refers to the dissociation rate of aparticular antibody-antigen interaction. The term “KD”, as used herein,is intended to refer to the dissociation constant, which is obtainedfrom the ratio of Kd to Ka (i.e., Kd/Ka) and is expressed as a molarconcentration (M). KD values for antibodies can be determined usingmethods well established in the art.

“Label” or a “detectable moiety” as used herein, refers broadly to acomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, chemical, or other physical means.

“Low stringency,” “medium stringency,” “high stringency,” or “very highstringency conditions,” as used herein, refers broadly to conditions fornucleic acid hybridization and washing. Guidance for performinghybridization reactions can be found in Ausubel, et al. (2002) ShortProtocols in Molecular Biology (5^(th) Ed.) John Wiley & Sons, NY.Exemplary specific hybridization conditions include but are not limitedto: (1) low stringency hybridization conditions in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by two washes in0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes canbe increased to 55° C. for low stringency conditions); (2) mediumstringency hybridization conditions in 6×SSC at about 45° C., followedby one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; (3) highstringency hybridization conditions in 6×SSC at about 45° C., followedby one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and (4) very highstringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C.

“Mammal,” as used herein, refers broadly to any and all warm-bloodedvertebrate animals of the class Mammalia, including humans,characterized by a covering of hair on the skin and, in the female,milk-producing mammary glands for nourishing the young. Examples ofmammals include but are not limited to alpacas, armadillos, capybaras,cats, camels, chimpanzees, chinchillas, cattle, dogs, goats, gorillas,hamsters, horses, humans, lemurs, llamas, mice, non-human primates,pigs, rats, sheep, shrews, squirrels, tapirs, and voles. Mammals includebut are not limited to bovine, canine, equine, feline, murine, ovine,porcine, primate, and rodent species. Mammal also includes any and allthose listed on the Mammal Species of the World maintained by theNational Museum of Natural History, Smithsonian Institution inWashington D.C.

“Naturally-occurring nucleic acid molecule,” as used herein, refersbroadly to refers to an RNA or DNA molecule having a nucleotide sequencethat occurs in nature (e.g., encodes a natural protein).

“Nucleic acid” or “nucleic acid sequence,” as used herein, refersbroadly to a deoxy-ribonucleotide or ribonucleotide oligonucleotide ineither single- or double-stranded form. The term encompasses nucleicacids, i.e., oligonucleotides, containing known analogs of naturalnucleotides. The term also encompasses nucleic-acid-like structures withsynthetic backbones. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions) andcomplementary sequences, as well as the sequence explicitly indicated.The term nucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

“Oligomerization domain”, as used herein, refers broadly to a domainthat when attached to a VISTA extracellular domain or fragment thereof,facilitates oligomerization. Said oligomerization domains compriseself-associating α-helices, for example, leucine zippers, that can befurther stabilized by additional disulfide bonds. The domains aredesigned to be compatible with vectorial folding across a membrane, aprocess thought to facilitate in vivo folding of the polypeptide into afunctional binding protein. Examples thereof are known in the art andinclude by way of example coiled GCN4, and COMP.

The α-helical coiled coil is probably the most widespread subunitoligomerization motif found in proteins. Accordingly, coiled coilsfulfill a variety of different functions. In several families oftranscriptional activators, for example, short leucine zippers play animportant role in positioning the DNA-binding regions on the DNA.Ellenberger, et al. (1992) Cell 71: 1223-1237. Coiled coils are alsoused to form oligomers of intermediate filament proteins. Coiled-coilproteins furthermore appear to play an important role in both vesicleand viral membrane fusion. Skehel and Wiley (1998) Cell 95: 871-874. Inboth cases hydrophobic sequences, embedded in the membranes to be fused,are located at the same end of the rod-shaped complex composed of abundle of long α-helices. This molecular arrangement is believed tocause close membrane apposition as the complexes are assembled formembrane fusion. The coiled coil is often used to controloligomerization. It is found in many types of proteins, includingtranscription factors include, but not limited to GCN4, viral fusionpeptides, SNARE complexes and certain tRNA synthetases, among others.Very long coiled coils are found in proteins such as tropomyosin,intermediate filaments and spindle-pole-body components. Coiled coilsinvolve a number of α-helices that are supercoiled around each other ina highly organized manner that associate in a parallel or anantiparallel orientation. Although dimers and trimers are the mostcommon. The helices may be from the same or from different proteins. Thecoiled-coil is formed by component helices coming together to bury theirhydrophobic seams. As the hydrophobic seams twist around each helix, sothe helices also twist to coil around each other, burying thehydrophobic seams and forming a supercoil. It is the characteristicinterdigitation of side chains between neighbouring helices, known asknobs-into-holes packing, that defines the structure as a coiled coil.The helices do not have to run in the same direction for this type ofinteraction to occur, although parallel conformation is more commonAntiparallel conformation is very rare in trimers and unknown inpentamers, but more common in intramolecular dimers, where the twohelices are often connected by a short loop. In the extracellular space,the heterotrimeric coiled-coil protein laminin plays an important rolein the formation of basement membranes. Other examples are thethrombospondins and cartilage oligomeric matrix protein (COMP) in whichthree (thrombospondins 1 and 2) or five (thrombospondins 3, 4 and COMP)chains are connected. The molecules have a flower bouquet-likeappearance, and the reason for their oligomeric structure is probablythe multivalent interaction of the C-terminal domains with cellularreceptors. The yeast transcriptional activator GCN4 is 1 of over 30identified eukaryotic proteins containing the basic region leucinezipper (bZIP) DNA-binding motif. Ellenberger, et al. (1992) Cell 71:1223-1237. The bZIP dimer is a pair of continuous alpha helices thatform a parallel coiled-coil over their carboxy-terminal 34 residues andgradually diverge toward their amino termini to pass through the majorgroove of the DNA binding site. The coiled-coil dimerization interfaceis oriented almost perpendicular to the DNA axis, giving the complex theappearance of the letter T. bZIP contains a 4-3 heptad repeat ofhydrophobic and nonpolar residues that pack together in a parallelalpha-helical coiled-coil. Ellenberger, et al. (1992) Cell 71:1223-1237. The stability of the dimer results from the side-by-sidepacking of leucines and nonpolar residues in positions a and d of theheptad repeat, as well as a limited number of intra- and interhelicalsalt bridges, shown in a crystal structure of the GCN4 leucine zipperpeptide. Ellenberger, et al. (1992) Cell 71: 1223-1237. Another exampleis CMP (matrilin-1) isolated from bovine tracheal cartilage as ahomotrimer of subunits of Mr 52,000 (Paulsson & Heinegard (1981) BiochemJ. 197: 367-375), where each subunit consists of a vWFA1 module, asingle EGF domain, a vWFA2 module and a coiled coil domain spanning fiveheptads. Kiss, et al. (1989) J. Biol. Chem. 264:8126-8134; Hauser andPaulsson (1994) J. Biol. Chem. 269: 25747-25753. Electron microscopy ofpurified CMP showed a bouquet-like trimer structure in which eachsubunit forms an ellipsoid emerging from a common point corresponding tothe coiled coil. Hauser and Paulsson (1994) J. Biol. Chem. 269:25747-25753. The coiled coil domain in matrilin-1 has been extensivelystudied. The trimeric structure is retained after complete reduction ofinterchain disulfide bonds under non-denaturing conditions. Hauser andPaulsson (1994) J. Biol. Chem. 269: 25747-25753. Yet another example isCartilage Oligomeric Matrix Protein (COMP). A non-collagenousglycoprotein, COMP, was first identified in cartilage. Hedbom, et al.(1992) J. Biol. Chem. 267:6132-6136. The protein is a 524 kDahomopentamer of five subunits which consists of an N-terminal heptadrepeat region (cc) followed by four epidermal growth factor (EGF)-likedomains (EF), seven calcium-binding domains (T3) and a C-terminalglobular domain (TC). According to this domain organization, COMPbelongs to the family of thrombospondins. Heptad repeats (abcdefg)_(n)with preferentially hydrophobic residues at positions a and dform-helical coiled-coil domains. Cohen and Parry (1994) Science 263:488-489. Recently, the recombinant five-stranded coiled-coil domain ofCOMP (COMPcc) was crystallized and its structure was solved at 0.2 nmresolution. Malashkevich, et al. (1996) Science 274: 761-765.

“Operatively linked”, as used herein, refers broadly to when two DNAfragments are joined such that the amino acid sequences encoded by thetwo DNA fragments remain in-frame.

“Paratope,” as used herein, refers broadly to the part of an antibodywhich recognizes an antigen (e.g., the antigen-binding site of anantibody.) Paratopes may be a small region (e.g., 15-22 amino acids) ofthe antibody's Fv region and may contain parts of the antibody's heavyand light chains. See Goldsby, et al. Antigens (Chapter 3) Immunology(5^(th) Ed.) New York: W.H. Freeman and Company, pages 57-75.

“Patient,” as used herein, refers broadly to any animal who is in needof treatment either to alleviate a disease state or to prevent theoccurrence or reoccurrence of a disease state. Also, “Patient” as usedherein, refers broadly to any animal who has risk factors, a history ofdisease, susceptibility, symptoms, signs, was previously diagnosed, isat risk for, or is a member of a patient population for a disease. Thepatient may be a clinical patient such as a human or a veterinarypatient such as a companion, domesticated, livestock, exotic, or zooanimal. The term “subject” may be used interchangeably with the term“patient.”

“Polypeptide,” “peptide” and “protein,” are used interchangeably andrefer broadly to a polymer of amino acid residues. The terms apply toamino acid polymers in which one or more amino acid residue is an analogor mimetic of a corresponding naturally occurring amino acid, as well asto naturally occurring amino acid polymers. The terms apply to aminoacid polymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer. Polypeptides can be modified, e.g., by theaddition of carbohydrate residues to form glycoproteins. The terms“polypeptide,” “peptide” and “protein” include glycoproteins, as well asnon-glycoproteins.

“Promoter,” as used herein, refers broadly to an array of nucleic acidsequences that direct transcription of a nucleic acid. As used herein, apromoter includes necessary nucleic acid sequences near the start siteof transcription, such as, in the case of a polymerase II type promoter,a TATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation.

“Prophylactically effective amount,” as used herein, refers broadly tothe amount of a compound that, when administered to a patient forprophylaxis of a disease or prevention of the reoccurrence of a disease,is sufficient to effect such prophylaxis for the disease orreoccurrence. The prophylactically effective amount may be an amounteffective to prevent the incidence of signs and/or symptoms. The“prophylactically effective amount” may vary depending on the diseaseand its severity and the age, weight, medical history, predisposition toconditions, preexisting conditions, of the patient to be treated.

“Prophylaxis,” as used herein, refers broadly to a course of therapywhere signs and/or symptoms are not present in the patient, are inremission, or were previously present in a patient. Prophylaxis includespreventing disease occurring subsequent to treatment of a disease in apatient. Further, prevention includes treating patients who maypotentially develop the disease, especially patients who are susceptibleto the disease (e.g., members of a patent population, those with riskfactors, or at risk for developing the disease).

“Recombinant” as used herein, refers broadly with reference to aproduct, e.g., to a cell, or nucleic acid, protein, or vector, indicatesthat the cell, nucleic acid, protein or vector, has been modified by theintroduction of a heterologous nucleic acid or protein or the alterationof a native nucleic acid or protein, or that the cell is derived from acell so modified. Thus, for example, recombinant cells express genesthat are not found within the native (non-recombinant) form of the cellor express native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

“Signal sequence” or “signal peptide,” as used herein, refers broadly toa peptide containing about 15 or more amino acids which occurs at theN-terminus of secretory and membrane bound polypeptides and whichcontains a large number of hydrophobic amino acid residues. For example,a signal sequence contains at least about 10-30 amino acid residues,preferably about 15-25 amino acid residues, more preferably about 18-20amino acid residues, and even more preferably about 19 amino acidresidues, and has at least about 35-65%, preferably about 38-50%, andmore preferably about 40-45% hydrophobic amino acid residues (e.g.,Valine, Leucine, Isoleucine or Phenylalanine). A “signal sequence,” alsoreferred to in the art as a “signal peptide,” serves to direct apolypeptide containing such a sequence to a lipid bilayer, and iscleaved in secreted and membrane bound polypeptides.

“Specifically (or selectively) binds” to an antibody or “specifically(or selectively) immunoreactive with,” or “specifically interacts orbinds,” as used herein, refers broadly to a protein or peptide (or otherepitope), refers, in some embodiments, to a binding reaction that isdeterminative of the presence of the protein in a heterogeneouspopulation of proteins and other biologics. For example, underdesignated immunoassay conditions, the specified antibodies bind to aparticular protein at least two times greater than the background(non-specific signal) and do not substantially bind in a significantamount to other proteins present in the sample. Typically a specific orselective reaction will be at least twice background signal or noise andmore typically more than about 10 to 100 times background.

“Specifically hybridizable” and “complementary” as used herein, referbroadly to a nucleic acid can form hydrogen bond(s) with another nucleicacid sequence by either traditional Watson-Crick or othernon-traditional types. The binding free energy for a nucleic acidmolecule with its complementary sequence is sufficient to allow therelevant function of the nucleic acid to proceed, e.g., RNAi activity.Determination of binding free energies for nucleic acid molecules iswell known in the art. See, e.g., Turner, et al. (1987) CSH Symp. Quant.Biol. LII: 123-33; Frier, et al. (1986) PNAS 83: 9373-77; Turner, et al.(1987) J. Am. Chem. Soc. 109: 3783-85. A percent complementarityindicates the percentage of contiguous residues in a nucleic acidmolecule that can form hydrogen bonds (e.g., Watson-Crick base pairing)with a second nucleic acid sequence (e.g., about at least 5, 6, 7, 8, 9,10 out of 10 being about at least 50%, 60%, 70%, 80%, 90%, and 100%complementary, inclusive). “Perfectly complementary” or 100%complementarity refers broadly all of the contiguous residues of anucleic acid sequence hydrogen bonding with the same number ofcontiguous residues in a second nucleic acid sequence. “Substantialcomplementarity” refers to polynucleotide strands exhibiting about atleast 90% complementarity, excluding regions of the polynucleotidestrands, such as overhangs, that are selected so as to benoncomplementary. Specific binding requires a sufficient degree ofcomplementarity to avoid non-specific binding of the oligomeric compoundto non-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, or in the case of in vitro assays,under conditions in which the assays are performed. The non-targetsequences typically may differ by at least 5 nucleotides.

“Signs” of disease, as used herein, refers broadly to any abnormalityindicative of disease, discoverable on examination of the patient; anobjective indication of disease, in contrast to a symptom, which is asubjective indication of disease.

“Solid support,” “support,” and “substrate,” as used herein, refersbroadly to any material that provides a solid or semi-solid structurewith which another material can be attached including but not limited tosmooth supports (e.g., metal, glass, plastic, silicon, and ceramicsurfaces) as well as textured and porous materials.

“Subjects” as used herein, refers broadly to anyone suitable to betreated according to the present invention include, but are not limitedto, avian and mammalian subjects, and are preferably mammalian. Anymammalian subject in need of being treated according to the presentinvention is suitable. Human subjects of both genders and at any stageof development (i.e., neonate, infant, juvenile, adolescent, adult) canbe treated according to the present invention. The present invention mayalso be carried out on animal subjects, particularly mammalian subjectssuch as mice, rats, dogs, cats, cattle, goats, sheep, and horses forveterinary purposes, and for drug screening and drug developmentpurposes. “Subjects” is used interchangeably with “patients.”

“Substantially free of chemical precursors or other chemicals,” as usedherein, refers broadly to preparations of VISTA protein in which theprotein is separated from chemical precursors or other chemicals whichare involved in the synthesis of the protein. In one embodiment, thelanguage “substantially free of chemical precursors or other chemicals”includes preparations of VISTA protein having less than about 30% (bydry weight) of chemical precursors or non-VISTA chemicals, morepreferably less than about 20% chemical precursors or non-VISTAchemicals, still more preferably less than about 10% chemical precursorsor non-VISTA chemicals, and most preferably less than about 5% chemicalprecursors or non-VISTA (PD-L3) chemicals.

“Symptoms” of disease as used herein, refers broadly to any morbidphenomenon or departure from the normal in structure, function, orsensation, experienced by the patient and indicative of disease.

“T cell,” as used herein, refers broadly to CD4+ T cells and CD8+ Tcells. The term T cell also includes both T helper 1 type T cells and Thelper 2 type T cells.

“Treg cell” (sometimes also referred to as suppressor T cells) as usedherein refers to a subpopulation of T cells which modulate the immunesystem and maintain tolerance to self-antigens and can abrogateautoimmune diseases. Foxp3+CD4+CD25+ regulatory T cells (Tregs) arecritical in maintaining peripheral tolerance under normal physiologicalconditions, and suppress anti-tumour immune responses in cancer.

“Therapy,” “therapeutic,” “treating,” or “treatment”, as used herein,refers broadly to treating a disease, arresting, or reducing thedevelopment of the disease or its clinical symptoms, and/or relievingthe disease, causing regression of the disease or its clinical symptoms.Therapy encompasses prophylaxis, treatment, remedy, reduction,alleviation, and/or providing relief from a disease, signs, and/orsymptoms of a disease. Therapy encompasses an alleviation of signsand/or symptoms in patients with ongoing disease signs and/or symptoms(e.g., inflammation, pain). Therapy also encompasses “prophylaxis”. Theterm “reduced”, for purpose of therapy, refers broadly to the clinicalsignificant reduction in signs and/or symptoms. Therapy includestreating relapses or recurrent signs and/or symptoms (e.g.,inflammation, pain). Therapy encompasses but is not limited toprecluding the appearance of signs and/or symptoms anytime as well asreducing existing signs and/or symptoms and eliminating existing signsand/or symptoms. Therapy includes treating chronic disease(“maintenance”) and acute disease. For example, treatment includestreating or preventing relapses or the recurrence of signs and/orsymptoms (e.g., inflammation, pain).

“Transmembrane domain,” as used herein, refers broadly to an amino acidsequence of about 15 amino acid residues in length which spans theplasma membrane. More preferably, a transmembrane domain includes aboutat least 20, 25, 30, 35, 40, or 45 amino acid residues and spans theplasma membrane. Transmembrane domains are rich in hydrophobic residues,and typically have an alpha-helical structure. In an embodiment, atleast 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of atransmembrane domain are hydrophobic, e.g., leucines, isoleucines,tyrosines, or tryptophans. Transmembrane domains are described in, forexample, Zagotta, et al. (1996) Annu. Rev. Neurosci. 19:235-263.

“Transgenic animal,” as used herein, refers broadly to a non-humananimal, preferably a mammal, more preferably a mouse, in which one ormore of the cells of the animal includes a “transgene”. The term“transgene” refers to exogenous DNA which is integrated into the genomeof a cell from which a transgenic animal develops and which remains inthe genome of the mature animal, for example directing the expression ofan encoded gene product in one or more cell types or tissues of thetransgenic animal.

“Tumor,” as used herein, refers broadly to at least one cell or cellmass in the form of a tissue neoformation, in particular in the form ofa spontaneous, autonomous and irreversible excess growth, which is moreor less disinhibited, of endogenous tissue, which growth is as a ruleassociated with the more or less pronounced loss of specific cell andtissue functions. This cell or cell mass is not effectively inhibited,in regard to its growth, by itself or by the regulatory mechanisms ofthe host organism, e.g., colorectal cancer, melanoma or carcinoma. Tumorantigens not only include antigens present in or on the malignant cellsthemselves, but also include antigens present on the stromal supportingtissue of tumors including endothelial cells and other blood vesselcomponents.

“Unresponsiveness,” as used herein, refers broadly to refractivity ofimmune cells to stimulation, e.g., stimulation via an activatingreceptor or a cytokine. Unresponsiveness can occur, e.g., because ofexposure to immunosuppressants or high doses of antigen.

“Variable region” or “VR,” as used herein, refers broadly to the domainswithin each pair of light and heavy chains in an antibody that areinvolved directly in binding the antibody to the antigen. Each heavychain has at one end a variable domain (V_(H)) followed by a number ofconstant domains. Each light chain has a variable domain (V_(L)) at oneend and a constant domain at its other end; the constant domain of thelight chain is aligned with the first constant domain of the heavychain, and the light chain variable domain is aligned with the variabledomain of the heavy chain.

“Vector,” as used herein, refers broadly to a nucleic acid moleculecapable of transporting another nucleic acid molecule to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Vectors are referred to herein as “recombinant expression vectors” orsimply “expression vectors”. In general, expression vectors of utilityin recombinant DNA techniques are often in the form of plasmids. In thepresent specification, “plasmid” and “vector” may be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions. The techniques and procedures are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification. See, e.g., Sambrook,et al. (2001) Molec. Cloning: Lab. Manual [3^(rd) Ed] Cold Spring HarborLaboratory Press. Standard techniques may be used for recombinant DNA,oligonucleotide synthesis, and tissue culture, and transformation (e.g.,electroporation, lipofection). Enzymatic reactions and purificationtechniques may be performed according to manufacturer's specificationsor as commonly accomplished in the art or as described herein.

The nomenclatures utilized in connection with, and the laboratoryprocedures and techniques of, analytical chemistry, synthetic organicchemistry, and medicinal and pharmaceutical chemistry described hereinare those well known and commonly used in the art. Standard techniquesmay be used for chemical syntheses, chemical analyses, pharmaceuticalpreparation, formulation, and delivery, and treatment of patients.

VISTA

This application relates to a novel, structurally-distinct,Ig-superfamily inhibitory ligand designated as V-regionImmunoglobulin-containing Suppressor of T cell Activation (VISTA) orPD-L3 that is selectively expressed on hematopoietic cells. Theextracellular domain bears homology to the B7 family ligand PD-L1, andlike PD-L1, VISTA has a profound impact on immunity. However, unlikePD-L1, VISTA is selectively expressed within the hematopoieticcompartment. Expression is most prominent on myeloid antigen-presentingcells (APCs), although expression on CD4+ T cells, CD8⁺ T cells andhigher expression on a subset of Foxp3+ regulatory T cells (Treg) isalso of significant interest. A soluble VISTA-Ig fusion protein, orVISTA expression on APCs, potently inhibits in vitro T cellproliferation, cytokine production and induces Foxp3 expression in Tcells. Conversely, a newly developed anti-VISTA monoclonal antibodyinterfered with VISTA-induced immune suppression of T cell responses byVISTA+ APCs in vitro. Furthermore, in vivo anti-VISTA intensified thedevelopment of the T cell mediated autoimmune disease experimentalallergic encephalomyelitis (EAE), and facilitated the development of aprotective, tumor-specific immune response with subsequent tumorremission. Initial studies of VISTA −/− mice are revealing earlyindications of spontaneous inflammatory disease, and their ultimatepathologic fate will be determined. Unlike all other PD-Ligand-relatedmolecules (e.g., B7-H3, H4, H6), VISTA is selectively expressed inhematopoietic cells, together with its profound suppressive activitiesand unique structural features, illustrates that VISTA is a novel,functionally non-redundant, central negative regulator of immunity,whose expression is primarily T cell and myeloid-restricted. See WO2011/120013.

The best characterized costimulatory ligands are B7.1 and B7.2 and theybelong to the Ig superfamily which consists of many critical immuneregulators, such as the B7 family ligands and receptors. Ig superfamilymembers are expressed on professional antigen-presenting cells (APCs),and their receptors are CD28 and CTLA-4. CD28 is expressed by naïve andactivated T cells and is critical for optimal T-cell activation. Incontrast, CTLA-4 is induced following T-cell activation and inhibitsT-cell activation by binding to B7.1/B7.2, impairing CD28-mediatedcostimulation. B7.1 and B7.2 knockout (KO) mice are impaired in adaptiveimmune response, whereas CTLA-4 KO mice cannot adequately controlinflammation and develop systemic autoimmune diseases. Over time the B7family ligands have expanded to include costimulatory ligands such asB7-H2 (ICOS Ligand) and B7-H3, and coinhibitory ligands such as B7-H1(PD-L1), B7-DC (PD-L2), B7-H4 (B7S1 or B7x), and B7-H6. Accordingly,additional CD28 family receptors have been identified. ICOS is expressedon activated T cells and binds to B7-H2. ICOS is a positiveco-regulator, important for T-cell activation, differentiation andfunction. On the other hand, Programmed Death 1 (PD-1) negativelyregulates T cell responses. PD-1 KO mice developed lupus-like autoimmunedisease, or T dilated cardiomyopathy. In contrast to VISTA, the twoinhibitory B7 family ligands, PD-L1 and PD-L2, have distinct expressionpatterns. PD-L2 is inducibly expressed on DCs and macrophages, whereasPD-L1 is broadly expressed on both hematopoietic cells andnonhematopoietic cell types. Consistent with the immune-suppressive roleof PD-1 receptor, studies using PD-L1−/− and PD-L2−/− mice have shownthat both ligands have overlapping roles in inhibiting T-cellproliferation and cytokine production. PD-L1 deficiency enhances diseaseprogression in both the non-obese diabetic (NOD) model of autoimmunediabetes and the murine model of multiple sclerosis (experimentalautoimmune encephalomyelitis (EAE). PD-L1−/− T cells produce elevatedlevels of the proinflammatory cytokines in both disease models. Inaddition, studies in NOD mice have demonstrated that the tissueexpression of PD-L1 (i.e., within pancreas) uniquely contributes to itscapacity of regionally controlling inflammation. PD-L1 is also highlyexpressed on placental syncytiotrophoblasts, which critically controlthe maternal immune responses to allogeneic fetus.

Anti-CTLA-4 antibodies show an enhanced therapeutic benefit in murinemodels and clinical trials of melanoma. Mice vaccinated with B16-GM-CSF(Gvax) promote the rejection of B16 melanomas when combined withantibody blockade of CTLA-4. Antibodies to PD-1 as well as PD-L1 alsodocument enhanced anti-tumor immunity and host survival in a wide rangeof murine tumor models. Finally, although CTLA-4 and PD-1 belong to thesame family of co-inhibitory molecules, evidence suggests they usedistinct nonredundant mechanisms to inhibit T-cell activation, and thereis synergy in the ability of anti-CTLA-4 and anti-PD-1/L1 to enhancehost survival in murine melanoma when used in combination.

The immunoglobulin (Ig) superfamily consists of many critical immuneregulators, including the B7 family ligands and receptors. VISTA is anovel and structurally distinct Ig superfamily inhibitory ligand, whoseextracellular domain bears homology to the B7 family ligand PD-L1. Thismolecule is designated V-domain Ig suppressor of T cell activation(VISTA). VISTA is primarily expressed on hematopoietic cells, and VISTAexpression is highly regulated on myeloid antigen-presenting cells(APCs) and T cells. A soluble VISTA-Ig fusion protein or VISTAexpression on APCs inhibits T cell proliferation and cytokine productionin vitro. A VISTA-specific monoclonal antibody interferes withVISTA-induced suppression of T cell responses by VISTA-expressing APCsin vitro. Furthermore, anti-VISTA treatment exacerbates the developmentof the T cell-mediated autoimmune disease experimental autoimmuneencephalomyelitis in mice. Finally, VISTA over expression on tumor cellsinterferes with protective antitumor immunity in vivo in mice. Thesefindings show that VISTA, a novel immunoregulatory molecule, hasfunctional activities that are nonredundant with other Ig superfamilymembers and may play a role in the development of autoimmunity andimmune surveillance in cancer. See Wang, et al. (2011) The Journal ofExperimental Medicine 208(3): 577-92.

Human VISTA (PD-L3) or VISTA was identified as an upregulated moleculein a T cell transcriptional profiling screen. Our characterization of anidentical 930 bp gene product recovered from a murine CD4⁺ T-cell cDNAlibrary confirmed the size and sequence. Silico-sequence and structuralanalysis predicts a type I transmembrane protein of 309 amino acids uponmaturation. Its extracellular domain contains a single extracellularIg-V domain of 136 amino acids, which is linked to a 23-amino acid stalkregion, a 21-residue transmembrane segment, and a 97-amino acidcytoplasmic domain. The cytoplasmic tail of VISTA does not contain anysignaling domains. A BLAST sequence search with the VISTA Ig-V domainidentified PD-L1 of the B7 family as the closest evolutionarily relatedprotein with a borderline significant e-value score. A structure basedsequence alignment of VISTA with the B7 family members PD-L1, PD-L2,B7-H3, and B7-H4 highlights several amino acids that are systematicallyconserved in all Ig-V domain proteins.

The expression of VISTA appears to be selectively expressed in thehematopoietic compartment and this protein is highly expressed on maturemyeloid cells (CD11b^(bright)), with lower levels of expression on CD4⁺T cells, T^(reg) and CD8⁺ T cells. Soluble VISTA proteins, e.g., solubleVISTA-Ig fusion protein, or VISTA expression on APCs, suppresses invitro CD4⁺ and CD8⁺ T cell proliferation and cytokine production. It isalso observed that anti-VISTA antibodies, e.g., an anti-VISTA monoclonalantibody (13F3) blocked VISTA-induced suppression of T cell responses byVISTA⁺ APCs in vitro. Also, it has been discovered that an anti-VISTAmonoclonal antibody exacerbated EAE and increased the frequency ofencephalitogenic Th17s in vivo. Still further, the inventors suprisinglydiscovered that an anti-VISTA monoclonal antibody induces tumorremission in multiple murine tumor models. VISTA expression on myeloidderived suppressor cells (MDSC) in these models is extremely high,suggesting that VISTA⁺ MDSC suppress tumor specific immunity VISTAexerts immunosuppressive activities on T cells both in vitro and invivo, in mouse and in human (in vitro only) and is an important mediatorin controlling the development of autoimmunity and the immune responsesto cancer. Specifically, the data show that VISTA is a new member of theIg superfamily and contains an Ig-V domain with distant sequencesimilarity to PD-L1. A VISTA-Ig fusion protein or when over expressed onartificial APCs VISTA inhibits both mouse and human CD4+ and CD8+ T cellproliferation and cytokine production. Further, VISTA expression onmyeloid APCs is inhibitory for T cell responses in vitro.

VISTA expression on MDSC in the tumor microenvironment is extremelyhigh. Phenotypic and functional analysis of many cell surface moleculespreviously suggested to be involved in MDSC-mediated suppression of Tcells: CD115, CD124, CD80, PD-L1, and PD-L2 were expressed by MDSC butwith no differences in the levels of their expression or proportion ofpositive cells were found between MDSC and cells from tumor-free micethat lack immune suppressive activity. Therefore, VISTA is the primaryB7 negative regulator on MDSCs.

Antibody-Mediated VISTA Blockade Induces Protective Immunity to anAutologous Tumor.

VISTA is a dominant, negative immune regulatory molecule on MDSCs thatinterferes with the development of protective anti-tumor immunity.Therefore, blocking the activity of this molecule with anti-VISTAantibodies may be used to induce protective anti-tumor immunity inmammals (e.g., humans).

Methods of using soluble VISTA proteins, e.g., fusion proteins andmultimeric VISTA proteins comprising multiple copies of the VISTAextracellular domain or a fragment thereof, and VISTA binding agents,e.g., small molecules and antibodies or fragments thereof, which bind ormodulate (agonize or antagonize) the activity of VISTA as immunemodulators and for the treatment of different cancers, e.g., bladder,ovarian and lymphoma, autoimmune disease, allergy, infection andinflammatory conditions, e.g. multiple sclerosis and arthritis.

VISTA is a novel inhibitory ligand, which extracellular Ig-V domainbears homology to the two known B7 family ligands Programmed DeathLigand 1 and 2 (PD-L1 and PD-L2) and exhibits unique sequence featuresand distinctive expression patterns in vitro and in vivo on subsets ofAPCs and T cells, (which distinguishes PD-L3 or VISTA from other B7family ligands). VISTA has a functional impact on CD4⁺ and CD8⁺ T cellproliferation and differentiation (suppresses CD4⁺ and CD8⁺ T cellproliferation, as well as cytokine production). Based on its expressionpattern and inhibitory impact on T cells, PD-L3 or VISTA apparentlyfunctions as a regulatory ligand that negatively regulates T cellresponses during cognate interactions between T cells and myeloidderived APCs.

Although VISTA (PD-L3) appears to be a member of the B7 family ofligands, unlike other B7 family ligands, this molecule contains only anIg-V domain without an Ig-C domain, and is phylogenically closer to theB7 family receptor Programmed Death-1 (PD-1). Based thereon, VISTA(PD-L3), and agonists or antagonists specific thereto can be used toregulate T cell activation and differentiation, and more broadly tomodulate the regulatory network that controls immune responses. Inparticular VISTA (PD-L3) proteins and VISTA (PD-L3) agonists orantagonists, preferably antibodies specific to VISTA (PD-L3) are usefulin modulating immune responses in autoimmunity, inflammatory responsesand diseases, allergy, cancer, infectious disease and transplantation.

Anergy in T cells (as opposed to unresponsiveness) is characterized bylack of cytokine production, e.g., IL-2. T cell anergy occurs when Tcells are exposed to antigen and receive a first signal (a T cellreceptor or CD-3 mediated signal) in the absence of a second signal (acostimulatory signal). Under these conditions, reexposure of the cellsto the same antigen (even if reexposure occurs in the presence of acostimulatory molecule) results in failure to produce cytokines and,thus, failure to proliferate. Anergic T cells can, however, mountresponses to unrelated antigens and can proliferate if cultured withcytokines (e.g., IL-2). For example, T cell anergy can also be observedby the lack of IL-2 production by T lymphocytes as measured by ELISA orby a proliferation assay using an indicator cell line. Alternatively, areporter gene construct can be used. For example, anergic T cells failto initiate IL-2 gene transcription induced by a heterologous promoterunder the control of the 5′ IL-2 gene enhancer or by a multimer of theAP1 sequence that can be found within the enhancer. Kang, et al. (1992)Science 257: 1134.

A VISTA (PD-L3) molecule of the present invention is identified based onthe presence of a “extracellular domain” in the polypeptide orcorresponding nucleic acid molecule. In another embodiment, a VISTA(PD-L3) molecule of the present invention is identified based on thepresence of a “cytoplasmic domain” in the polypeptide or correspondingnucleic acid molecule.

Methods for modulating an immune cell response by contacting an immunecell in vitro or in vivo with a VISTA protein, or binding agent specificthereto, in the presence of a primary signal so that a response of theimmune cell is modulated. (Interaction of VISTA or a modulator thereoftransmits a signal to immune cells, regulating immune responses. VISTA(PD-L3) protein is expressed at high levels on myeloid antigenpresenting cells, including myeloid dendritic cells (DCs) andmacrophages, and at lower densities on CD4+ and CD8+ T cells. Uponimmune activation, VISTA (PD-L3) expression is upregulated on myeloidAPCs, but downregulated on CD4+ T cells). Therefore, the VISTA (PD-L3)nucleic acids and polypeptides of the present invention, and agonists orantagonists thereof are useful, e.g., in modulating the immune response.

As used interchangeably herein, “VISTA (PD-L3) activity”, “biologicalactivity of VISTA (PD-L3)” or “functional activity of VISTA (PD-L3)”,refers to an activity exerted by a VISTA (PD-L3) protein, polypeptide ornucleic acid molecule on a VISTA (PD-L3)-responsive cell or tissue, oron a VISTA (PD-L3) polypeptide binding partner, as determined in vivo,or in vitro, according to standard techniques. These activities includemodulating CD4+ and CD8+ T cell proliferation and cytokine production.In another embodiment, a VISTA (PD-L3) activity is a direct activity,such as an association with a VISTA (PD-L3) binding partner. As usedherein, a “target molecule” or “binding partner” is a molecule withwhich a VISTA (PD-L3) polypeptide binds or interacts in nature, i.e.,expressed on a T cell, such that VISTA (PD-L3)-mediated function isachieved. Alternatively, a VISTA (PD-L3) activity is an indirectactivity, such as a cellular signaling activity mediated by the VISTA(PD-L3) polypeptide. The biological activities of VISTA (PD-L3) aredescribed herein. For example, the VISTA (PD-L3) polypeptides and VISTA(PD-L3) agonists or antagonists of the present invention can have one ormore of the following activities: (1) suppresses or promotes CD4+ andCD8+ T cell proliferation, (2) suppresses or promotes cytokineproduction (3) functions as a regulatory ligand that negativelyregulates T cell responses during cognate interactions between T cellsand myeloid derived APCs (4) negatively regulates CD4+ T cell responsesby suppressing early TCR activation and arresting cell division, butwith minimum direct impact on apoptosis, (5) suppresses or promotesantigen-specific T cell activation during cognate interactions betweenAPCs and T cells and/or (6) suppresses or promotes T cell-mediatedimmune responses; (7) modulate activation of immune cells, e.g., Tlymphocytes, and (8) modulate the immune response, e.g., inflammatoryimmune response of an organism, e.g., a mouse or human organism.

Isolated VISTA (PD-L3) proteins and polypeptides that modulate one ormore VISTA (PD-L3) activities. These polypeptides will include VISTA(PD-L3) polypeptides having one or more of the following domains: asignal peptide domain, an IgV domain, an extracellular domain, atransmembrane domain, and a cytoplasmic domain, and, preferably, a VISTA(PD-L3) activity.

Modulation of a costimulatory signal may result in modulation ofeffector function of an immune cell. Thus, the term “VISTA activity”includes the ability of a VISTA polypeptide to bind its natural bindingpartner(s), the ability to modulate immune cell costimulatory orinhibitory signals, and the ability to modulate the immune response.

Modulation of an inhibitory signal in an immune cell results inmodulation of proliferation of and/or cytokine secretion by an immunecell. For example, the family of VISTA (PD-L3) polypeptides of thepresent invention preferably comprises least one “signal peptidedomain”. As described infra a signal sequence was identified in theamino acid sequence of native human VISTA (PD-L3) and was alsoidentified in the amino acid sequence of native mouse VISTA (PD-L3).

Stimulation of VISTA (PD-L3) activity is desirable in situations inwhich VISTA (PD-L3) is abnormally downregulated and/or in whichincreased VISTA (PD-L3) activity is likely to have a beneficial effect.Likewise, inhibition of VISTA (PD-L3) activity is desirable insituations in which VISTA (PD-L3) is abnormally upregulated and/or inwhich decreased VISTA (PD-L3) activity is likely to have a beneficialeffect. Exemplary agents for use in downmodulating VISTA (PD-L3) (i.e.,VISTA (PD-L3) antagonists) include, e.g., antisense nucleic acidmolecules, antibodies that recognize and block VISTA (PD-L3),combinations of antibodies that recognize and block VISTA (PD-L3) andantibodies that recognize and block VISTA (PD-L3) counter receptors, andcompounds that block the interaction of VISTA (PD-L3) with its naturallyoccurring binding partner(s) on an immune cell (e.g., soluble,monovalent VISTA (PD-L3) molecules; soluble forms of VISTA (PD-L3)molecules that do not bind Fc receptors on antigen presenting cells;soluble forms of VISTA (PD-L3) binding partners; and compoundsidentified in the subject screening assays). Exemplary agents for use inupmodulating VISTA (PD-L3) (i.e., VISTA (PD-L3) agonists) include, e.g.,nucleic acid molecules encoding VISTA (PD-L3) polypeptides, multivalentforms of VISTA (PD-L3), compounds that increase the expression of VISTA(PD-L3), compounds that enhance the interaction of VISTA (PD-L3) withits naturally occurring binding partners and cells that express VISTA(PD-L3).

Depending upon the form of the VISTA (PD-L3) molecule that binds to areceptor, a signal can be either transmitted (e.g., form of a VISTA(PD-L3) molecule that results in crosslinking of the receptor or by asoluble form of VISTA (PD-L3) that binds to Fc receptors on antigenpresenting cells) or inhibited (e.g., by a soluble, monovalent form of aVISTA (PD-L3) molecule or a soluble form of VISTA (PD-L3) that isaltered using methods known in the art such that it does not bind to Fcreceptors on antigen presenting cells), e.g., by competing withactivating forms of VISTA (PD-L3) molecules for binding to the receptor.However, there are instances in which a soluble molecule can bestimulatory. The effects of the various modulatory agents can be easilydemonstrated using routine screening assays as described herein.

Downregulation of Immune Responses

Upregulating the inhibitory function of a VISTA (PD-L3) polypeptide maybe used to downregulate immune responses. Downregulation can be in theform of inhibiting or blocking an immune response already in progress,or may involve preventing the induction of an immune response. Thefunctions of activated immune cells can be inhibited by downregulatingimmune cell responses or by inducing specific anergy in immune cells, orboth. For example, VISTA (PD-L3) may bind to an inhibitory receptor,forms of VISTA (PD-L3) that bind to the inhibitory receptor, e.g.,multivalent VISTA (PD-L3) on a cell surface, can be used to downmodulatethe immune response. An activating antibody may be used to stimulateVISTA (PD-L3) activity is a bispecific antibody. For example, such anantibody can comprise a VISTA (PD-L3) binding site and another bindingsite which targets a cell surface receptor on an immune cell, e.g., a Tcell, a B cell, or a myeloid cell. Such an antibody, in addition tocomprising a VISTA (PD-L3) binding site, can further comprise a bindingsite which binds to a B cell antigen receptor, a T cell antigenreceptor, or an Fc receptor, in order to target the molecule to aspecific cell population. Selection of this second antigen for thebispecific antibody provides flexibility in selection of cell populationto be targeted for inhibition. Agents that promote a VISTA (PD-L3)activity or which enhance the interaction of VISTA (PD-L3) with itsnatural binding partners (e.g., VISTA (PD-L3) activating antibodies orVISTA (PD-L3) activating small molecules) can be identified by theirability to inhibit immune cell proliferation and/or effector function,or to induce anergy when added to an in vitro assay. For example, cellscan be cultured in the presence of an agent that stimulates signaltransduction via an activating receptor. A number of art-recognizedreadouts of cell activation can be employed to measure, e.g., cellproliferation or effector function (e.g., antibody production, cytokineproduction, phagocytosis) in the presence of the activating agent. Theability of a test agent to block this activation can be readilydetermined by measuring the ability of the agent to effect a decrease inproliferation or effector function being measured. In one embodiment, atlow antigen concentrations, VISTA (PD-L3) immune cell interactionsinhibit strong B7-CD28 signals. In another embodiment, at high antigenconcentrations, VISTA (PD-L3) immune cell interactions may reducecytokine production but not inhibit T cell proliferation. Accordingly,the ability of a test compound to block activation can be determined bymeasuring cytokine production and/or proliferation at differentconcentrations of antigen.

Tolerance may be induced against specific antigens by co-administeringan antigen with a VISTA (PD-L3) agonist. For example, tolerance may beinduced to specific polypeptides Immune responses to allergens orforeign polypeptides to which an immune response is undesirable can beinhibited. For example, patients that receive Factor VIII frequentlygenerate antibodies against this clotting factor. Co-administration ofan agent that stimulates VISTA (PD-L3) activity or interaction with itsnatural binding partner, with recombinant factor VIII (or physicallylinking VISTA (PD-L3) to Factor VIII, e.g., by cross-linking) can resultin immune response downmodulation.

A VISTA (PD-L3) agonist and another agent that can block activity ofcostimulatory receptors on an immune cell can be used to downmodulateimmune responses. Exemplary molecules include: agonists forms of otherPD ligands, soluble forms of CTLA-4, anti-B7-1 antibodies, anti-B7-2antibodies, or combinations thereof. Alternatively, two separatepeptides (for example, a VISTA (PD-L3) polypeptide with blocking formsof B7-2 and/or B7-1 polypeptides), or a combination of antibodies (e.g.,activating antibodies against a VISTA (PD-L3) polypeptide with blockinganti-B7-2 and/or anti-B7-1 monoclonal antibodies) can be combined as asingle composition or administered separately (simultaneously orsequentially) to downregulate immune cell mediated immune responses in asubject. Furthermore, a therapeutically active amount of one or morepeptides having a VISTA (PD-L3) polypeptide activity, along with one ormore polypeptides having B7-1 and/or B7-1 activity, can be used inconjunction with other downmodulating reagents to influence immuneresponses. Examples of other immunomodulating reagents includeantibodies that block a costimulatory signal (e.g., against CD28 orICOS), antibodies that activate an inhibitory signal via CTLA4, and/orantibodies against other immune cell markers (e.g., against CD40, CD40ligand, or cytokines), fusion proteins (e.g., CTLA4-Fc or PD-1-Fc), andimmunosuppressive drugs (e.g., rapamycin, cyclosporine A, or FK506). TheVISTA (PD-L3) polypeptides may also be useful in the construction oftherapeutic agents which block immune cell function by destruction ofcells. For example, portions of a VISTA (PD-L3) polypeptide can belinked to a toxin to make a cytotoxic agent capable of triggering thedestruction of cells to which it binds.

Infusion of one or a combination of such cytotoxic agents (e.g., VISTA(PD-L3) ricin (alone or in combination with PD-L1-ricin), into a patientmay result in the death of immune cells, particularly in light of thefact that activated immune cells that express higher amounts of VISTA(PD-L3) binding partners. For example, because PD-1 is induced on thesurface of activated lymphocytes, a VISTA (PD-L3) polypeptide can beused to target the depletion of these specific cells by Fc-R dependentmechanisms or by ablation by conjugating a cytotoxic drug (e.g., ricin,saporin, or calicheamicin) to the VISTA (PD-L3) polypeptide to killcells that express a receptor for VISTA. A toxin can be conjugated to ananti-VISTA (PD-L3) antibody in order to target for death VISTA(PD-L3)-expressing antigen-presenting cell. In a further embodiment, theVISTA (PD-L3)-antibody-toxin can be a bispecific antibody. Suchbispecific antibodies are useful for targeting a specific cellpopulation, e.g., using a marker found only on a certain type of cell,e.g., B lymphocytes, monocytes, dendritic cells, or Langerhans cells.Downregulating immune responses by activating VISTA (PD-L3) activity orthe VISTA (PD-L3)-immune cell interaction (and thus stimulating thenegative signaling function of VISTA (PD-L3)) is useful indownmodulating the immune response, e.g., in situations of tissue, skinand organ transplantation, in graft-versus-host disease (GVHD), orallergies, or in autoimmune diseases such as systemic lupuserythematosus and multiple sclerosis. For example, blockage of immunecell function results in reduced tissue destruction in tissuetransplantation. Typically, in tissue transplants, rejection of thetransplant is initiated through its recognition as foreign by immunecells, followed by an immune reaction that destroys the transplant. Theadministration of a molecule which promotes the activity of VISTA(PD-L3) or the interaction of VISTA (PD-L3) with its natural bindingpartner(s), on immune cells (such as a soluble, multimeric form of aVISTA (PD-L3) polypeptide) alone or in conjunction with anotherdownmodulatory agent prior to or at the time of transplantation caninhibit the generation of a costimulatory signal. Moreover, promotion ofVISTA (PD-L3) activity may also be sufficient to anergize the immunecells, thereby inducing tolerance in a subject.

To achieve sufficient immunosuppression or tolerance in a subject, itmay also be desirable to block the costimulatory function of othermolecules. For example, it may be desirable to block the function ofB7-1 and B7-2 by administering a soluble form of a combination ofpeptides having an activity of each of these antigens or blockingantibodies against these antigens (separately or together in a singlecomposition) prior to or at the time of transplantation. Alternatively,it may be desirable to promote inhibitory activity of VISTA (PD-L3) andinhibit a costimulatory activity of B7-1 and/or B7-2. Otherdownmodulatory agents that can be used in connection with thedownmodulatory methods of the invention include, for example, agentsthat transmit an inhibitory signal via CTLA4, soluble forms of CTLA4,antibodies that activate an inhibitory signal via CTLA4, blockingantibodies against other immune cell markers, or soluble forms of otherreceptor ligand pairs (e.g., agents that disrupt the interaction betweenCD40 and CD40 ligand (e.g., anti CD40 ligand antibodies)), antibodiesagainst cytokines, or immunosuppressive drugs. For example, activatingVISTA (PD-L3) activity or the interaction of VISTA (PD-L3) with itsnatural binding partner(s), is useful in treating autoimmune disease.Many autoimmune disorders are the result of inappropriate activation ofimmune cells that are reactive against self tissue and which promote theproduction of cytokines and autoantibodies involved in the pathology ofthe diseases. Preventing the activation of autoreactive immune cells mayreduce or eliminate disease symptoms. Administration of agents thatpromote activity of VISTA (PD-L3) (PD-L3) or VISTA interaction with itsnatural binding partner(s), may induce antigen-specific tolerance ofautoreactive immune cells which could lead to long-term relief from thedisease. Additionally, co-administration of agents which blockcostimulation of immune cells by disrupting receptor-ligand interactionsof B7 molecules with costimulatory receptors may be useful in inhibitingimmune cell activation to prevent production of autoantibodies orcytokines which may be involved in the disease process. The efficacy ofreagents in preventing or alleviating autoimmune disorders can bedetermined using a number of well-characterized animal models of humanautoimmune diseases. Examples include murine experimental autoimmuneencephalitis, systemic lupus erythematosus in MRL/lpr/lpr mice or NZBhybrid mice, murine autoimmune collagen arthritis, diabetes mellitus inNOD mice and BB rats, and murine experimental myasthenia gravis. SeePaul ed., Fundamental Immunology, Raven Press, New York, 1989, pages840-856.

Inhibition of immune cell activation is useful therapeutically in thetreatment of allergies and allergic reactions, e.g., by inhibiting IgEproduction. An agent that promotes VISTA (PD-L3) activity or VISTA(PD-L3) interaction with its natural binding partner(s) can beadministered to an allergic subject to inhibit immune cell-mediatedallergic responses in the subject. Stimulation VISTA (PD-L3) activity orinteraction with its natural binding partner(s), can be accompanied byexposure to allergen in conjunction with appropriate MHC molecules.Allergic reactions can be systemic or local in nature, depending on theroute of entry of the allergen and the pattern of deposition of IgE onmast cells or basophils. Thus, immune cell-mediated allergic responsescan be inhibited locally or systemically by administration of an agentthat promotes VISTA (PD-L3) activity or VISTA (PD-L3)-immune cellinteractions.

Downregulation of an immune response via stimulation of VISTA (PD-L3)activity or VISTA (PD-L3) interaction with its natural bindingpartner(s), may also be useful in treating an autoimmune attack ofautologous tissues. Thus, conditions that are caused or exacerbated byautoimmune attack (e.g., heart disease, myocardial infarction oratherosclerosis) may be ameliorated or improved by increasing VISTA(PD-L3) activity or VISTA (PD-L3) biding to its natural binding partner.It is therefore within the scope of the invention to modulate conditionsexacerbated by autoimmune attack, such as autoimmune disorders (as wellas conditions such as heart disease, myocardial infarction, andatherosclerosis) by stimulating VISTA (PD-L3) activity or VISTA (PD-L3)interaction with its counter receptor.

Upregulation of Immune Responses

Inhibition of VISTA (PD-L3) activity or VISTA (PD-L3) interaction withits natural binding partner(s), as a means of upregulating immuneresponses is also useful in therapy. Upregulation of immune responsescan be in the form of enhancing an existing immune response or elicitingan initial immune response. For example, enhancing an immune responsethrough inhibition of VISTA (PD-L3) activity is useful in cases ofinfections with microbes, e.g., bacteria, viruses, or parasites, or incases of immunosuppression. For example, an agent that inhibits VISTA(PD-L3) activity, e.g., a non-activating antibody (i.e., a blockingantibody) against VISTA (PD-L3), or a soluble form of VISTA (PD-L3), istherapeutically useful in situations where upregulation of antibody andcell-mediated responses, resulting in more rapid or thorough clearanceof a virus, bacterium, or parasite, would be beneficial. Theseconditions include viral skin diseases such as Herpes or shingles, inwhich case such an agent can be delivered topically to the skin. Inaddition, systemic viral diseases such as influenza, the common cold,and encephalitis might be alleviated by the administration of suchagents systemically. In certain instances, it may be desirable tofurther administer other agents that upregulate immune responses, forexample, forms of B7 family members that transduce signals viacostimulatory receptors, in order further augment the immune response.

Immune responses may be enhanced in an infected patient by removingimmune cells from the patient, contacting immune cells in vitro with anagent that inhibits the VISTA (PD-L3) activity or VISTA (PD-L3)interaction with its natural binding partner(s), and reintroducing thein vitro-stimulated immune cells into the patient. In anotherembodiment, a method of enhancing immune responses involves isolatinginfected cells from a patient, e.g., virally infected cells,transfecting them with a nucleic acid molecule encoding a form of VISTA(PD-L3) that cannot bind its natural binding partner(s), such that thecells express all or a portion of the VISTA (PD-L3) molecule on theirsurface, and reintroducing the transfected cells into the patient. Thetransfected cells may be capable of preventing an inhibitory signal to,and thereby activating, immune cells in vivo.

A agent that inhibits VISTA (PD-L3) activity or VISTA (PD-L3)interaction with its natural binding partner(s), can be usedprophylactically in vaccines against various polypeptides, e.g.,polypeptides derived from pathogens. Immunity against a pathogen, e.g.,a virus, can be induced by vaccinating with a viral polypeptide alongwith an agent that inhibits VISTA (PD-L3) activity, in an appropriateadjuvant. Alternately, a vector comprising genes which encode for both apathogenic antigen and a form of VISTA (PD-L3) that blocks VISTA (PD-L3)interaction with immune cells can be used for vaccination. Nucleic acidvaccines can be administered by a variety of means, for example, byinjection (e.g., intramuscular, intradermal, or the biolistic injectionof DNA-coated gold particles into the epidermis with a gene gun thatuses a particle accelerator or a compressed gas to inject the particlesinto the skin. Haynes, et al. (1996) J. Biotechnol. 44:37.Alternatively, nucleic acid vaccines can be administered by non-invasivemeans. For example, pure or lipid-formulated DNA can be delivered to therespiratory system or targeted elsewhere, e.g., Peyers patches by oraldelivery of DNA. Schubbert (1997) Proc Natl. Acad. Sci. USA 94: 961.Attenuated microorganisms can be used for delivery to mucosal surfaces.Sizemore et al. (1995) Science 270:29.

The antigen in the vaccine may be a self-antigen. Such a vaccine isuseful in the modulation of tolerance in an organism. Immunization witha self antigen and an agent that blocks VISTA (PD-L3) activity or VISTA(PD-L3) interaction with its natural binding partner can break tolerance(i.e., interfere with tolerance of a self antigen). Such a vaccine mayalso include adjuvants such as alum or cytokines (e.g., GM-CSF, IL-12,B7-1, or B7-2). In one embodiment, an agent which inhibits VISTA (PD-L3)activity or VISTA (PD-L3) interaction with its natural bindingpartner(s), can be administered with class I MHC polypeptides by, forexample, a cell transfected to coexpress a VISTA (PD-L3) polypeptide orblocking antibody and MHC class I α chain polypeptide and β2microglobulin to result in activation of T cells and provide immunityfrom infection. For example, viral pathogens for which vaccines areuseful include: hepatitis B, hepatitis C, Epstein-Barr virus,cytomegalovirus, HIV-1, HIV-2, tuberculosis, malaria andschistosomiasis.

Inhibition of VISTA (PD-L3) activity or VISTA (PD-L3) interaction withits natural binding partner(s), can be useful in the treatment of tumorimmunity Tumor cells (e.g., colorectal cancer, sarcoma, melanoma,lymphoma, leukemia, neuroblastoma, or carcinoma) can be transfected witha nucleic acid molecule that inhibits VISTA (PD-L3) activity. Thesemolecules can be, e.g., nucleic acid molecules which are antisense toVISTA (PD-L3), or can encode non-activating anti-VISTA (PD-L3)antibodies. These molecules can also be the variable region of ananti-VISTA (PD-L3) antibody. If desired, the tumor cells can also betransfected with other polypeptides which activate costimulation (e.g.,B7-1 or B7-2). The transfected tumor cells are returned to the patient,which results in inhibition (e.g., local inhibition) of VISTA (PD-L3)activity Alternatively, gene therapy techniques can be used to target atumor cell for transfection in vivo.

Stimulation of an immune response to tumor cells can also be achieved byinhibiting VISTA (PD-L3) activity or VISTA (PD-L3) interaction with itsnatural binding partner(s), by treating a patient with an agent thatinhibits VISTA (PD-L3) activity or VISTA (PD-L3) interaction with itsnatural binding partner(s). Preferred examples of such agents include,e.g., antisense nucleic acid molecules, antibodies that recognize andblock VISTA (PD-L3), and compounds that block the interaction of VISTA(PD-L3) with its naturally occurring binding partner(s) on an immunecell (e.g., soluble, monovalent VISTA (PD-L3) molecules; soluble formsof VISTA (PD-L3) molecules that do not bind to Fc receptors on antigenpresenting cells; soluble forms of VISTA (PD-L3) binding partner(s); andcompounds identified in the subject screening assays). In addition,tumor cells which lack MHC class I or MHC class II molecules, or whichfail to express sufficient amounts of MHC class I or MHC class IImolecules, can be transfected with nucleic acid encoding all or aportion of (e.g., a cytoplasmic-domain truncated portion) of an MHCclass I α chain polypeptide and beta2 microglobulin polypeptide or anMHC class II α chain polypeptide and an MHC class II β chain polypeptideto thereby express MHC class I or MHC class II polypeptides on the cellsurface. Expression of the appropriate class I or class II MHC inconjunction with an VISTA (PD-L3) inhibiting polypeptide or antisensenucleic acid induces a T cell mediated immune response against thetransfected tumor cell. Optionally, a gene encoding an antisenseconstruct which blocks expression of an MHC class II-associatedpolypeptide, such as the invariant chain, can also be cotransfected witha DNA encoding a VISTA (PD-L3) inhibiting polypeptide or antisensenucleic acid to promote presentation of tumor associated antigens andinduce tumor specific immunity. Expression of B7-1 by B7-negative murinetumor cells has been shown to induce T cell mediated specific immunityaccompanied by tumor rejection and prolonged protection to tumorchallenge in mice. Chen, et al. (1992) Cell 71: 1093-1102; Townsend &Allison (1993) Science 259: 368-370; Baskar, et al. (1993) Proc Natl.Acad. Sci. 90: 5687-5690. Thus, the induction of an immune cell-mediatedimmune response in a human subject can be sufficient to overcometumor-specific tolerance in the subject. In another embodiment, theimmune response can be stimulated by the inhibition of VISTA (PD-L3)activity or VISTA (PD-L3) interaction with its natural bindingpartner(s), such that preexisting tolerance is overcome. For example,immune responses against antigens to which a subject cannot mount asignificant immune response, e.g., tumor-specific antigens, can beinduced by administering an agent that inhibits the activity of VISTA(PD-L3) activity or the ability of VISTA (PD-L3) to bind to its naturalbinding partner, can be used as adjuvants to boost responses to foreignantigens in the process of active immunization.

Immune cells may be obtained from a subject and cultured ex vivo in thepresence of an agent that that inhibits VISTA (PD-L3) activity or VISTA(PD-L3) interaction with its natural binding partner(s), to expand thepopulation of immune cells. In a further embodiment the immune cells arethen administered to a subject. Immune cells can be stimulated toproliferate in vitro by, for example, providing the immune cells with aprimary activation signal and a costimulatory signal, as is known in theart. Various forms of VISTA (PD-L3) polypeptides or agents that inhibitVISTA (PD-L3) activity can also be used to costimulate proliferation ofimmune cells. In one embodiment, immune cells are cultured ex vivoaccording to the methods described in WO 94/29436. The costimulatorymolecule can be soluble, attached to a cell membrane or attached to asolid surface, such as a bead.

In performing any of the methods described herein, it is within thescope of the invention to upregulate an immune response by administeringone or more additional agents. For example, the use of other agentsknown to stimulate the immune response, such as cytokines, adjuvants, orstimulatory forms of costimulatory molecules or their ligands can beused in conjunction with an agent that inhibits VISTA (PD-L3) activityor VISTA (PD-L3) interaction with its natural binding partner(s).

Identification of Cytokines Modulated by Modulation of VISTA (PD-L3)Activity or VISTA (PD-L3)-Interactions with its Counter Receptor on TCells

The VISTA (PD-L3) molecules described herein may be used to identifycytokines which are produced by or whose production is enhanced orinhibited in immune cells in response to modulation of VISTA (PD-L3)activity or VISTA (PD-L3) interaction with its natural bindingpartner(s), Immune cells may be suboptimally stimulated in vitro with aprimary activation signal, for example, T cells can be stimulated withphorbol ester, anti-CD3 antibody or preferably, antigen, in associationwith an MHC class II molecule, and given a costimulatory signal, e.g.,by a stimulatory form of B7 family antigen, for instance by a celltransfected with nucleic acid encoding a B7 polypeptide and expressingthe peptide on its surface, or by a soluble, stimulatory form of thepeptide. The cells can then be contacted with cells expressing VISTA(PD-L3) (e.g., antibodies against VISTA (PD-L3) Known cytokines releasedinto the media can be identified by ELISA or by the ability of anantibody which blocks the cytokine to inhibit immune cell proliferationor proliferation of other cell types that are induced by the cytokine.For example, an IL-4 ELISA kit is available from Genzyme (Cambridge,Mass.), as is an IL-7 blocking antibody. Blocking antibodies againstIL-9 and IL-12 are available from Genetics Institute (Cambridge, Mass.).The effect of stimulating or blocking VISTA (PD-L3) activity or theinteraction of VISTA (PD-L3) and its binding partner(s) on the cytokineprofile can then be determined. As noted supra and shown in the examplesVISTA (PD-L3) apparently suppresses the expression of IL-2 and gammainterferon by immune cells.

An in vitro immune cell costimulation assay as described above can alsobe used in a method for identifying novel cytokines which can bemodulated by modulation of VISTA (PD-L3) activity. For example, wherestimulation of the CD28/CTLA4 pathway seems to enhance IL-2 secretion,stimulation of the ICOS pathway seems to enhance IL-10 secretion.Hutloff, et al. (1999) Nature 397: 263. If a particular activity inducedupon costimulation, e.g., immune cell proliferation, cannot be inhibitedby addition of blocking antibodies to known cytokines, the activity mayresult from the action of an unknown cytokine. Following costimulation,this cytokine can be purified from the media by conventional methods andits activity measured by its ability to induce immune cellproliferation.

To identify cytokines which may play a role the induction of tolerance,an in vitro T cell costimulation assay as described above can be used.In this case, T cells would be given the primary activation signal andcontacted with a selected cytokine, but would not be given thecostimulatory signal. After washing and resting the immune cells, thecells would be rechallenged with both a primary activation signal and acostimulatory signal. If the immune cells do not respond (e.g.,proliferate or produce cytokines) they have become tolerized and thecytokine has not prevented the induction of tolerance. However, if theimmune cells respond, induction of tolerance has been prevented by thecytokine. Those cytokines which are capable of preventing the inductionof tolerance can be targeted for blockage in vivo in conjunction withreagents which block B lymphocyte antigens as a more efficient means toinduce tolerance in transplant recipients or subjects with autoimmunediseases. For example, one could administer a cytokine blocking antibodyto a subject along with an agent that promotes VISTA (PD-L3) activity orVISTA (PD-L3) interaction with a binding partner.

Thus, to summarize a novel member of the Programmed Death Ligand (PDL)family has now been identified which is expressed by Treg cells. Thisnovel protein has been designated VISTA (PD-L3). The receptors of thisPD-L family are type I transmembrane proteins containing a single IgVdomain, while the ligands are type I transmembrane proteins expressingboth an IgV and an IgC extracellular domains. Like other members of thePDL family, VISTA (PD-L3) co-stimulates αCD3 proliferation of T cells invitro. In addition, the expression of VISTA (PD-L3) is increased in αCD3activated Treg and reduced in the presence of αGITR.

A second, TNF-like, protein has also been identified as beingupregulated upon αCD3/αGITR stimulation. This protein has beendesignated Treg-sTNF. These proteins may be involved incontact-dependent and paracrine suppression of immunity and thereforeare useful for modulating (e.g., inhibiting or stimulating) an immuneresponse and in the treatment of diseases and conditions involving Tregsignaling. For example, the VISTA (PD-L3) protein can be used as aco-stimulatory signal for stimulating or enhancing immune cellactivation. VISTA (PD-L3) proteins and VISTA (PD-L3) binding agents andVISTA (PD-L3) agonists and antagonists are especially useful in treatingimmune conditions wherein regulation of T cell immunity is desired,e.g., modulation of T cell activation, differentiation andproliferation, and in particular modulation of CD4+ and CD8+ T cellproliferation, cytokine production, and T cell responses during cognateinteractions between T cells and myeloid derived APCs.

VISTA and VISTA Conjugate Polypeptides

The invention provides VISTA and VISTA conjugate polypeptides. Theinventors surprisingly discovered that VISTA and VISTA conjugatepolypeptides act as negative immune modulators. Exemplary VISTApolypeptides are provided in SEQ ID NO: 2, 4, and 5. VISTA (PD-L3)molecules of the invention include at least one or more of the followingdomains: a signal peptide domain, an IgV domain, an extracellulardomain, a transmembrane domain, or a cytoplasmic domain. Isolatedpolypeptides of the present invention, preferably VISTA (PD-L3)polypeptides, may comprise an amino acid sequence sufficiently identicalto the amino acid sequence of SEQ ID NO: 2 or 4, or 5 or are encoded bya nucleotide sequence sufficiently identical to SEQ ID NO: 1 or 3 orfragment or complement thereof. As used herein, the term “sufficientlyidentical” refers to a first amino acid or nucleotide sequence whichcontains a sufficient or minimum number of identical or equivalent(e.g., an amino acid residue which has a similar side chain) amino acidresidues or nucleotides to a second amino acid or nucleotide sequencesuch that the first and second amino acid or nucleotide sequences sharecommon structural domains or motifs and/or a common functional activity.For example, amino acid or nucleotide sequences which share commonstructural domains have at least 30%, 40%, or 50% homology, preferably60% homology, more preferably 70-80%, and even more preferably 90-95%homology across the amino acid sequences of the domains and contain atleast one and preferably two structural domains or motifs, are definedherein as sufficiently identical. Furthermore, amino acid or nucleotidesequences which share at least 30%, 40%, or 50%, preferably 60%, morepreferably 70-80%, or 90-95% homology and share a common functionalactivity are defined herein as sufficiently identical. An extracellulardomain of the VISTA polypeptide may comprise an IgV domain and mayinclude a signal peptide domain. See FIGS. 1 and 23.

VISTA (PD-L3) polypeptides may have at least one extracellular domain,and one or more of a signal peptide domain, an IgV domain, antransmembrane domain, and a cytoplasmic domain, and are, preferably,encoded by a nucleic acid molecule having a nucleotide sequence whichhybridizes under stringent hybridization conditions to a nucleic acidmolecule comprising a complement of the nucleotide sequence of SEQ IDNO: 1 or 3 herein. The nucleotide and amino acid sequences sequence ofthe exemplified isolated human and murine VISTA (PD-L3) cDNA and thepredicted amino acid sequence of the human VISTA (PD-L3) polypeptide arecontained in the sequence listing herein.

A VISTA (PD-L3) polypeptide of the present invention may be identifiedbased on the presence of a “transmembrane domain”. The transmembranedomain region of PDL3 are identified herein. See e.g., FIGS. 1 and 23. AVISTA (PD-L3) molecule of the present invention may be identified basedon the absence of an “IgC domain” and the presence of an “IgV domain” inthe polypeptide or corresponding nucleic acid molecule. The amino acidresidues of the native human and murine VISTA (PD-L3) polypeptide,constituting the IgV domain can be seen in FIGS. 1 and 23. The presenceof an IgV domain is likely required for binding of VISTA (PD-L3) to itsnatural binding partner(s).

Nucleic acids encoding VISTA polypeptides may be modified using standardmolecular biological techniques that result in variants polypeptidescomprising at least one VISTA and VISTA conjugate including but notlimited to deletions, additions and substitutions in the amino acidsequence, that retain the specific antigenicity of the VISTA and VISTAconjugate (e.g., the VISTA polypeptides is bound by an anti-VISTAantibody). Additionally, variant polypeptides comprising at least oneVISTA polypeptide may also retain the antigenicity of the VISTApolypeptide (e.g., raise a specific immune response against the VISTApolypeptide and variant VISTA polypeptide, respectively, uponimmunization in a subject). The VISTA and VISTA conjugate polypeptidesmay be formulated with a pharmaceutical carrier to manufacture anantigen composition useful as a “cancer vaccine” (e.g., a pharmaceuticalcomposition that elicits a specific immune response against the VISTAand VISTA conjugate, that produces anti-tumor antibodies afterimmunization in a subject). The VISTA polypeptides and VISTA conjugatesdescribed herein may be used to treat autoimmune disorders andinflammatory diseases.

Polypeptide Derivatives and Analogs

It will be appreciated that polypeptides described herein may bedegradation products, synthetic peptides or recombinant peptides as wellas peptidomimetics, synthetic peptides, peptoids, and semipeptoids(e.g., peptide analogs, which may have, for example, modificationsrendering the peptides more stable while in a body or more capable ofpenetrating into cells.) Modifications of the VISTA and VISTA conjugatepolypeptides described herein include, but are not limited to N-terminusmodification, C-terminus modification, peptide bond modification (e.g.,CH₂—NH, CH₂—S, CH₂—S═O, O═C—NH, CH₂—O, CH₂—CH₂, S═C—NH, CH═CH or CF═CH),backbone modifications, and residue modification. Methods for preparingpeptidomimetic compounds are well known in the art. Martin, (2010)Quantitative Drug Design: A Critical Introduction [2^(nd) Ed.] CRCPress.

Peptide bonds (—CO—NH—) within the peptide may be substituted, forexample, by N-methylated bonds (—N(CH₃)—CO—), ester bonds(—C(R)H—C—O—O—C(R)—N—), ketomethylen bonds (—CO—CH2-), α-aza bonds(—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds(—CH₂—NH—), hydroxyethylene bonds (—CH(OH)—CH₂—), thioamide bonds(—CS—NH—), olefinic double bonds (—CH═CH—), retro amide bonds (—NH—CO—),peptide derivatives (—N(R)—CH₂—CO—), wherein R is the “normal” sidechain, naturally presented on the carbon atom. These modifications canoccur at any of the bonds along the peptide chain and even at several(2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted bysynthetic non-natural acid such as phenylglycine, TIC, naphthylelanine(Nol), ring-methylated derivatives of phenylalanine, halogenatedderivatives of phenylalanine or o-methyl-tyrosine. In addition to theabove, the polypeptides of the present invention may also include one ormore modified amino acids or one or more non-amino acid monomers (e.g.fatty acids, complex carbohydrates), for example, hydroxyproline,phosphoserine and phosphothreonine; and other unusual amino acidsincluding, but not limited to, 2-aminoadipic acid, hydroxylysine,isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, theterm “amino acid” includes both D- and L-amino acids.

Since the polypeptides of the present invention are preferably utilizedin therapeutics which requires the peptides to be in soluble form, thepolypeptides of the present invention may comprise one or morenon-natural or natural polar amino acids, including but not limited toserine and threonine which are capable of increasing peptide solubilitydue to their hydroxyl-containing side chain.

The polypeptides of the present invention may be in a linear form,although it will be appreciated that in cases may also be utilized.

The VISTA and VISTA conjugate polypeptides described herein may bepurified from cells that have been altered to express it (e.g.,recombinant). DNA sequences encoding the VISTA and VISTA conjugatepolypeptides may be inserted into an expression vector and thentransformed (or transfected) in an appropriate host cell and/orexpressed in a transgenic animal. The VISTA and VISTA conjugatepolypeptides so expressed may then be isolated by methods known in theart. See, e.g., Maniatis, et al. (2001) Molecular Cloning: A LaboratoryManual [3^(rd) Ed.] Cold Spring Harbor Laboratory Press.

The polypeptides of the present invention may be biochemicallysynthesized such as by using standard solid phase techniques. Thesemethods include exclusive solid phase synthesis, partial solid phasesynthesis methods, fragment condensation, classical solution synthesis.These methods are preferably used when the peptide is relatively short(i.e., 10 kDa) and/or when it cannot be produced by recombinanttechniques (i.e., not encoded by a nucleic acid sequence) and thereforeinvolves different chemistry. Solid phase peptide synthesis proceduresare well known in the art and further described by Stewart (1984) SolidPhase Peptide Syntheses [2^(nd) Ed.] Pierce Chemical Company andBenoiton (2005) Chemistry of Peptide Synthesis CRC Press. Syntheticpeptides may be purified by preparative high performance liquidchromatography and the composition of which may be confirmed via aminoacid sequencing. See Creighton (1992) [2^(nd) Ed.] Proteins, Structuresand Molecular Principles W.H. Freeman and Company; Aguilar (2004) [Ed.]HPLC of Peptides and Proteins: Methods and Protocols Humana Press;Simpson (2002) Protein Sequencing Protocols [2^(nd) Ed.] Humana Press.

In cases where large amounts of the polypeptides of the presentinvention are desired, the polypeptides of the present invention may begenerated using recombinant techniques such as described by Invitrogen(2002) “Guide to Baculovirus Expression Vector Systems (BEVs) and InsectCulture Techniques” Instruction Manual; Hatti-Kaul and Mattiasson (2003)[Eds] Isolation and Purification of Proteins; Ahmed (2004) Principlesand Reactions of Protein Extraction, Purification and CharacterizationCRC Press. Further recombinant techniques such as described by, forexample, Bitter, et al. (1987) Methods in Enzymol. 153: 516-544,Studier, et al. (1990) Methods in Enzymol. 185: 60-89, Brisson, et al.(1984) Nature 310: 511-514, Takamatsu, et al. (1987) EMBO J. 6: 307-311,Coruzzi, et al. (1984) EMBO J. 3: 1671-1680 and Brogli, et al. (1984)Science 224: 838-843, Gurley, et al. (1986) Mol. Cell. Biol. 6: 559-565and Weissbach & Weissbach (1988) Methods for Plant Molecular Biology,Academic Press, NY, Section VIII, pages 421-463.

Polypeptide Sequence Variants

For any VISTA and VISTA conjugate sequence described herein, furthercharacterization or optimization may be achieved by systematicallyeither adding or removing amino acid residues to generate longer orshorter peptides, and testing those and sequences generated by walking awindow of the longer or shorter size up or down the antigen from thatpoint. Coupling this approach to generating new candidate targets withtesting for effectiveness of antigenic molecules based on thosesequences in an immunogenicity assay, as known in the art or asdescribed herein, may lead to further manipulation of the antigen.Further still, such optimized sequences may be adjusted by, e.g., theaddition, deletions, or other mutations as known in the art and/ordiscussed herein to further optimize the VISTA and VISTA conjugate(e.g., increasing serum stability or circulating half-life, increasingthermal stability, enhancing delivery, enhance immunogenicity,increasing solubility, targeting to a particular in vivo location orcell type).

The VISTA and VISTA conjugate polypeptides described herein may compriseconservative substitution mutations, (i.e., the substitution of one ormore amino acids by similar amino acids). For example, conservativesubstitution refers to the substitution of an amino acid with anotherwithin the same general class, e.g., one acidic amino acid with anotheracidic amino acid, one basic amino acid with another basic amino acid,or one neutral amino acid by another neutral amino acid.

VISTA and VISTA conjugate polypeptide sequences may have at least about60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, or 100% sequence homology to any one or more ofthe polypeptide sequences of SEQ ID NO: 2, 4, or 5. More preferably, theinvention contemplates polypeptide sequences having at least about 95%sequence homology, even more preferably at least about 98% sequencehomology, and still more preferably at least about 99% sequence homologyto any one or more of the polypeptide sequences of VISTA and VISTAconjugate polypeptide sequences of SEQ ID NO: 2, 4, or 5. Methods fordetermining homology between amino acid sequences, as well as nucleicacid sequences, are well known to those of ordinary skill in the art.See, e.g., Nedelkov & Nelson (2006) New and Emerging ProteomicTechniques Humana Press.

Thus, a VISTA and VISTA conjugate polypeptide may have at least about80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with apolypeptide sequence. For example, a VISTA and VISTA conjugatepolypeptide may have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence homology with SEQ ID NO: 2, 4, or 5.

The term homology, or identity, is understood as meaning the number ofagreeing amino acids (identity) with other proteins, expressed inpercent. The identity is preferably determined by comparing a givensequence with other proteins with the aid of computer programs. Ifsequences which are compared with each other are different in length,the identity is to be determined in such a way that the number of aminoacids which the short sequence shares with the longer sequencedetermines the percentage identity. The identity can be determinedroutinely by means of known computer programs which are publiclyavailable such as, for example, ClustalW. Thompson, et al. (1994)Nucleic Acids Research 22: 4673-4680. ClustalW is publicly availablefrom the European Molecular Biology Laboratory and may be downloadedfrom various internet pages, inter alia the IGBMC (Institut de Génétiqueet de Biologie Moléculaire et Cellulaire) and the EBI and all mirroredEBI internet pages (European Bioinformatics Institute). If the ClustalWcomputer program Version 1.8 is used to determine the identity between,for example, the reference protein of the present application and otherproteins, the following parameters are to be set: KTUPLE=1, TOPDIAG=5,WINDOW=5, PAIRGAP=3, GAPOPEN=10, GAPEXTEND=0.05, GAPDIST=8, MAXDIV=40,MATRIX=GONNET, ENDGAPS(OFF), NOPGAP, NOHGAP. See also EuropeanBioinformatics Institute (EBI) toolbox available on-line and Smith(2002) Protein Sequencing Protocols [2^(nd) Ed.] Humana Press.

One possibility of finding similar sequences is to carry out sequencedatabase researches. Here, one or more sequences may be entered as whatis known as a query. This query sequence is then compared with sequencespresent in the selected databases using statistical computer programs.Such database queries (blast searches) are known to the skilled workerand may be carried out at different suppliers. If, for example, such adatabase query is carried out at the NCBI (National Center forBiotechnology Information), the standard settings for the respectivecomparison query should be used. For protein sequence comparisons(blastp), these settings are: Limit entrez=not activated; Filter=lowcomplexity activated; Expect value=10; word size=3; Matrix=BLOSUM62; Gapcosts: Existence=11, Extension=1. The result of such a query is, amongother parameters, the degree of identity between the query sequence andthe similar sequences found in the databases.

VISTA and VISTA conjugates include functional fragments of saidpolypeptides. A “functional fragment” of said polypeptide includes afragment of the gene or cDNA encoding said VISTA and VISTA conjugate,which fragment is capable of eliciting an immune response (e.g., humoralor cellular immune response.) Thus, for example, fragments of the VISTAand VISTA conjugate according to the invention which correspond to aminoacid residues that contribute to the immunogenicity of the antigen andwhich fragments may serve to function as antigens to elicit an immuneresponse (e.g., humoral or cellular immune response.) This aspect of theinvention also includes differentially spliced isoforms andtranscriptional starts of the polypeptides according to the invention.The polypeptides according to the invention also may comprise fragments,derivatives and allelic variants of the VISTA and VISTA conjugates.Methods and materials for making fragments of VISTA and VISTA conjugatepolypeptides are well known in the art. See, e.g., Maniatis, et al.(2001) Molecular Cloning: A Laboratory Manual [3^(rd) Ed.] Cold SpringHarbor Laboratory Press.

Variant VISTA and VISTA conjugate polypeptides may retain theirantigenic specificity to bind their respective antibodies (e.g., avariant VISTA polypeptide will be bound by an anti-VISTA antibody.)Fully antigenic variants may contain only conservative variations orvariations in non-critical residues or in non-critical regions.Antigenic variants may also contain substitution of similar amino acidsthat result in no change or an insignificant change in antigenicity.Alternatively, such substitutions may positively or negatively affectantigenicity to some degree. Non-antigenic variants typically containone or more non-conservative amino acid substitutions, deletions,insertions, inversions, or truncation or a substitution, insertion,inversion, or deletion in a critical residue or critical region of anepitope. Molecular biology and biochemistry techniques for modifyingVISTA and VISTA conjugate polypeptides while preserving specificantigenicity of the polypeptides for their respective antibodies arewell known in the art. See, e.g., Ho, et al. (1989) Gene 77(1): 51-59;Landt, et al. (1990) Gene 96(1): 125-128; Hopp & Woods (1991) Proc.Natl. Acad. Sci. USA 78(6): 3824-3828; Kolaskar & Tongaonkar (1990) FEBSLetters 276(1-2): 172-174; and Welling, et al. (1985) FEBS Letters188(2): 215-218.

Variants of the VISTA polypeptides which function as either VISTAagonists (mimetics) or as VISTA antagonists. Variants of the VISTApolypeptides can be generated by mutagenesis, e.g., discrete pointmutation or truncation of a VISTA polypeptide. An agonist of the VISTApolypeptides can retain substantially the same, or a subset, of thebiological activities of the naturally occurring form of a VISTApolypeptide. An antagonist of a VISTA polypeptide can inhibit one ormore of the activities of the naturally occurring form of the VISTApolypeptide by, for example, competitively modulating a VISTA-mediatedactivity of a VISTA polypeptide. Thus, specific biological effects canbe elicited by treatment with a variant of limited function. Forexample, a subject may be treated with a variant having a subset of thebiological activities of the naturally occurring form of the polypeptidehas fewer side effects in a subject relative to treatment with thenaturally occurring form of the VISTA polypeptide.

Variants of a VISTA polypeptide which function as either VISTA agonists(mimetics) or as VISTA antagonists may be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of a VISTApolypeptide for VISTA polypeptide agonist or antagonist activity.Diseases treatable with the subject VISTA (PD-L3) binding agents areidentified previously and include various inflammatory, autoimmune,cancer, allergic and infectious disorders. A particularly preferredindication is multiple sclerosis.

Peptidomimetics

In addition to VISTA polypeptides consisting only of naturally-occurringamino acids, VISTA peptidomimetics are also provided. Peptide analogsare commonly used in the pharmaceutical industry as non-peptide drugswith properties analogous to those of the template peptide. These typesof non-peptide compounds are termed “peptide mimetics” or“peptidomimetics” (Fauchere (1986) Adv. Drug Res. 15: 29; Advances inAmino Acid Mimetics and Peptidomimetics (Volume 2) Andrew Abell (Ed.)(1999) JAI Press, Inc. and Evans et al. (1987) J. Med. Chem 30: 1229)and are usually developed with the aid of computerized molecularmodeling. Peptide mimetics that are structurally similar totherapeutically useful peptides can be used to produce an equivalenttherapeutic or prophylactic effect. Generally, peptidomimetics arestructurally similar to a paradigm polypeptide (i.e., a polypeptide thathas a biological or pharmacological activity), such as human or mouseVISTA, but have one or more peptide linkages optionally replaced by alinkage selected from the group consisting of —CH₂NH—, —CH₂S—,—CH₂—CH₂—, —CH═CH— (cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—,by methods known in the art and further described in the followingreferences: Spatola in Chemistry and Biochemistry of Amino Acids,Peptides, and Proteins Weinstein, B., ed., Marcel Dekker, New York, p.267 (1983); Spatola, Vega Data (March 1983), Vol. 1, Issue 3, “PeptideBackbone Modifications”; Morley (1980) Trends. Pharm. Sci. pp. 463-468;Hudson, et al. (1979) Int. J. Pept. Prot. Res. 14:177-185 (—CH₂NH—,CH₂CH₂—); Spatola, et al. (1986) Life. Sci. 38:1243-1249 (—CH2-S); Hann,(1982) J. Chem. SoC Perkin. Trans. I 307-314 (—CH—CH—, cis and trans);Almquist, et al. (1980) J. Med. Chem. 23:1392-1398 (—COCH₂—);Jennings-White, et al. (1982) Tetrahedron Lett. 23:2533 (—COCH₂—);(—CH(OH)CH₂—); Holladay, et al. (1983) Tetrahedron. Lett. 24:4401-4404(—C(OH)CH₂—); and Hruby (1982) Life Sci. 31:189-199 (—CH₂—S—). Aparticularly preferred non-peptide linkage is —CH₂NH—. Such peptidemimetics may have significant advantages over polypeptide embodiments,including, for example: more economical production, greater chemicalstability, enhanced pharmacological properties (half-life, absorption,potency, efficacy), altered specificity (e.g., a broad-spectrum ofbiological activities), reduced antigenicity, and others. Labeling ofpeptidomimetics usually involves covalent attachment of one or morelabels, directly or through a spacer (e.g., an amide group), tonon-interfering position(s) on the peptidomimetic that are predicted byquantitative structure-activity data and/or molecular modeling. Suchnon-interfering positions generally are positions that do not formdirect contacts with the macromolecules(s) to which the peptidomimeticbinds to produce the therapeutic effect. Derivitization (e.g., labeling)of peptidomimetics should not substantially interfere with the desiredbiological or pharmacological activity of the peptidomimetic.

Systematic substitution of one or more amino acids of a VISTA amino acidsequence with a D-amino acid of the same type (e.g., D-lysine in placeof L-lysine) can be used to generate more stable peptides. In addition,constrained peptides comprising a VISTA amino acid sequence or asubstantially identical sequence variation can be generated by methodsknown in the art (Rizo and Gierasch (1992) Annu. Rev. Biochem. 61:387);for example, by adding internal cysteine residues capable of formingintramolecular disulfide bridges which cyclize the peptide. The aminoacid sequences of the VISTA polypeptides identified herein will enablethose of skill in the art to produce polypeptides corresponding to VISTApeptide sequences and sequence variants thereof. Such polypeptides canbe produced in prokaryotic or eukaryotic host cells by expression ofpolynucleotides encoding a VISTA peptide sequence, frequently as part ofa larger polypeptide. Alternatively, such peptides can be synthesized bychemical methods. Methods for expression of heterologous polypeptides inrecombinant hosts, chemical synthesis of polypeptides, and in vitrotranslation are well known in the art. Certain amino-terminal and/orcarboxy-terminal modifications and/or peptide extensions to the coresequence can provide advantageous physical, chemical, biochemical, andpharmacological properties, such as: enhanced stability, increasedpotency and/or efficacy, resistance to serum proteases, desirablepharmacokinetic properties, and others. Peptides can be usedtherapeutically to treat disease, e.g., by altering costimulation in apatient.

Amino acids that are essential for function may be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis. Cunningham, et al. (1989) Sci. 244: 1081-85. The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as epitope binding or in vitro ADCC activity. Sites thatare critical for ligand-receptor binding may also be determined bystructural analysis such as crystallography, nuclear magnetic resonance,or photoaffinity labeling. Smith, et al. (1992) J. Mol. Biol. 224:899-904; de Vos, et al. (1992) Sci. 255: 306-12.

For example, one class of substitutions is conserved amino acidsubstitutions. Such substitutions are those that substitute a givenamino acid in a VISTA and VISTA conjugate polypeptide with another aminoacid of like characteristics. Typically seen as conservativesubstitutions are the replacements, one for another, among the aliphaticamino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residuesSer and Thr, exchange of the acidic residues Asp and Glu, substitutionbetween the amide residues Asn and Gln, exchange of the basic residuesLys and Arg, replacements among the aromatic residues Phe, Tyr. Guidanceconcerning which amino acid changes are likely to be phenotypicallysilent is found in, for example, Bowie, et al. (1990) Sci. 247: 1306-10.Hence, one of ordinary skill in the art appreciates that the inventorspossess peptide variants without delineation of all the specificvariants. As to amino acid sequences, one of skill will recognize thatindividual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters, adds or deletesa single amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. Such conservativelymodified variants are in addition to and do not exclude polymorphicvariants, interspecies homologs, and alleles of the invention. See,e.g., Creighton (1992) Proteins: Structures and Molecular Properties[2^(nd) Ed.] W.H. Freeman.

Moreover, polypeptides often contain amino acids other than the twenty“naturally occurring” amino acids. Further, many amino acids, includingthe terminal amino acids, may be modified by natural processes, such asprocessing and other post-translational modifications, or by chemicalmodification techniques well known in the art. Known modificationsinclude, but are not limited to, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent crosslinks, formation of cystine, formation of pyroglutamate,formylation, g-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. See Creighton(1992) Proteins: Structure and Molecular Properties [2^(nd) Ed.] andLundblad (1995) Techniques in Protein Modification [1^(st) Ed.] Manydetailed reviews are available on this subject. See, e.g., Wold (1983)Posttranslational Covalent Modification of Proteins Acad. Press, NY;Seifter, et al. (1990) Meth. Enzymol. 182: 626-46; and Rattan, et al.(1992) Ann. NY Acad. Sci. 663: 48-62.

Fragments

A biologically active portion of a VISTA polypeptide includes a fragmentof a VISTA polypeptide which participates in an interaction between aVISTA molecule and a non-VISTA molecule, e.g., a natural ligand ofVISTA. Biologically active portions of a VISTA polypeptide includepeptides comprising amino acid sequences sufficiently identical to orderived from the amino acid sequence of the VISTA polypeptide, e.g., theamino acid sequence shown in SEQ ID NO: 2, 4 or 5, which include feweramino acids than the full length VISTA polypeptides, and exhibit atleast one activity of a VISTA polypeptide. Typically, biologicallyactive portions comprise a domain or motif with at least one activity ofthe VISTA polypeptide, e.g., modulating (suppressing) CD4 T cellproliferative responses to anti-CD3, suppression of the proliferativeresponse of cognate CD4 T cells in an antigen specific manner, effectson the expression of specific cytokines. A biologically active portionof a VISTA polypeptide can be a polypeptide which is, for example, 25,50, 75, 100, 125, 150, 175, 200, 225 or more amino acids in length.Biologically active portions of a VISTA polypeptide can be used astargets for developing agents which modulate a VISTA-mediated activity,e.g., immune cell activation.

A biologically active portion of a VISTA polypeptide may comprise atleast a portion of an extracellular domain. A biologically activeportion of a VISTA polypeptide may contain at least a portion of anextracellular domain (e.g., comprising an IgV), and one or more of thefollowing domains: a signal peptide domain, a transmembrane domain, or acytoplasmic domain. Moreover, other biologically active portions, inwhich other regions of the polypeptide are deleted, can be prepared byrecombinant techniques and evaluated for one or more of the functionalactivities of a native VISTA polypeptide.

The VISTA polypeptide may have the amino acid sequence shown in SEQ IDNO: 2, 4 or 5. The VISTA polypeptide may be substantially identical toSEQ ID NO: 2, 4 or 5, and retains the functional activity of thepolypeptide of SEQ ID NO: 2, 4 or 5, yet differs in amino acid sequencedue to natural allelic variation or mutagenesis, as described herein.

Fusion Proteins

Fusions comprising the VISTA and VISTA conjugate polypeptides are alsowithin the scope of the present invention. For example, the fusionprotein may be linked to a GST fusion protein in which the VISTA andVISTA conjugate polypeptide sequences are fused to the C-terminus of theGST sequences. Such fusion proteins may facilitate the purification ofthe recombinant VISTA and VISTA conjugate polypeptides. Alternatively,VISTA and VISTA conjugate polypeptides may be fused with a protein thatbinds B-cell follicles, thus initiating both a humoral immune responseand activation of T cells. Berney, et al. (1999) J. Exp. Med. 190:851-60. Alternatively, for example, the VISTA and VISTA conjugatepolypeptides may be genetically coupled with and anti-dendritic cellantibody to deliver the antigen to the immune system and stimulate acellular immune response. He, et al. (2004) Clin. Cancer Res. 10:1920-27. A chimeric or fusion protein of the invention may be producedby standard recombinant DNA techniques. For example, DNA fragmentscoding for the different polypeptide sequences are ligated togetherin-frame in accordance with conventional techniques, e.g., by employingblunt-ended or stagger-ended termini for ligation, restriction enzymedigestion to provide for appropriate termini, filling-in of cohesiveends as appropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. The fusion gene may be synthesized byconventional techniques including automated DNA synthesizers.

Fusion proteins may include C-terminal or N-terminal translocationsequences. Further, fusion proteins can comprise additional elements,e.g., for protein detection, purification, or other applications.Detection and purification facilitating domains including but notlimited to metal chelating peptides such as polyhistidine tracts,histidine-tryptophan modules, or other domains that allow purificationon immobilized metals; maltose binding protein; protein A domains thatallow purification on immobilized immunoglobulin; or the domain utilizedin the FLAG extension/affinity purification system (Immunex Corp,Seattle Wash.)

A fusion protein may be prepared from a protein of the invention byfusion with a portion of an immunoglobulin comprising a constant regionof an immunoglobulin. More preferably, the portion of the immunoglobulincomprises a heavy chain constant region which is optionally and morepreferably a human heavy chain constant region. The heavy chain constantregion is most preferably an IgG heavy chain constant region, andoptionally and most preferably is an Fc chain, most preferably an IgG Fcfragment that comprises CH2 and CH3 domains. Although any IgG subtypemay optionally be used, the IgG1 subtype is preferred. The Fc chain mayoptionally be a known or “wild type” Fc chain, or alternatively may bemutated. See, e.g., U.S. Patent Application Publication No.2006/0034852. The term “Fc chain” also optionally comprises any type ofFc fragment. Several of the specific amino acid residues that areinvolved in antibody constant region-mediated activity in the IgGsubclass have been identified. Inclusion, substitution or exclusion ofthese specific amino acids therefore allows for inclusion or exclusionof specific immunoglobulin constant region-mediated activity.Furthermore, specific changes may result in aglycosylation for exampleand/or other desired changes to the Fc chain. At least some changes mayoptionally be made to block a function of Fc which is considered to beundesirable, such as an undesirable immune system effect. SeeMcCafferty, et al. (2002) Antibody Engineering: A Practical Approach(Eds.) Oxford University Press.

The inclusion of a cleavable linker sequences such as Factor Xa (See,e.g., Ottavi, (1998) Biochimie 80: 289-93), subtilisin proteaserecognition motif (See, e.g., Polyak (1997) Protein Eng. 10: 615-19);enterokinase (Invitrogen, San Diego, Calif.), between the translocationdomain (for efficient plasma membrane expression) and the rest of thenewly translated polypeptide may be useful to facilitate purification.For example, one construct can include a polypeptide encoding a nucleicacid sequence linked to six histidine residues followed by athioredoxin, an enterokinase cleavage site (See, e.g., Williams (1995)Biochemistry 34: 1787-97), and an C-terminal translocation domain. Thehistidine residues facilitate detection and purification while theenterokinase cleavage site provides a means for purifying the desiredprotein(s) from the remainder of the fusion protein. Technologypertaining to vectors encoding fusion proteins and application of fusionproteins are well described in the scientific and patent literature.See, e.g., Kroll (1993) DNA Cell. Biol. 12: 441-53.

A fusion protein may be a GST-VISTA fusion protein in which the VISTAsequences are fused to the C-terminus of the GST sequences. Such fusionproteins can facilitate the purification of recombinant VISTA. Inanother embodiment, the fusion protein is a VISTA polypeptide containinga heterologous signal sequence at its N-terminus. In certain host cells(e.g., mammalian host cells), expression and/or secretion of VISTA canbe increased through use of a heterologous signal sequence. In anembodiment, the fusion protein is an Ig-VISTA fusion protein in whichthe VISTA sequences are fused to a portion of an Ig molecule. The Igportion of the fusion protein can include and immunoglobulin constantregion, e.g., a human Cγ1 domain or a Cγ4 domain (e.g., the hinge, CH2,and CH3 regions of human IgCγ1 or human IgCγ4 (see, e.g., U.S. Pat. Nos.5,116,964; 5,580,756; 5,844,095). A resulting fusion protein may havealtered VISTA solubility, binding affinity, stability and/or valency(i.e., the number of binding sites per molecule) and may increase theefficiency of protein purification.

Particularly preferred VISTA Ig fusion proteins include an extracellulardomain portion of VISTA coupled to an immunoglobulin constant region(e.g, the Fc region). The immunoglobulin constant region may containgenetic modifications which reduce or eliminate effector activityinherent in the immunoglobulin structure. For example, DNA encoding anextracellular portion of a VISTA polypeptide can be joined to DNAencoding the hinge, CH2, and CH3 regions of human IgGγ1 and/or IgGγ4modified by site-directed mutagenesis, e.g., as taught in WO 97/28267.The VISTA fusion proteins of the invention can be incorporated intopharmaceutical compositions and administered to a subject in vivo. TheVISTA fusion proteins can be used to affect the bioavailability of aVISTA binding partner. Use of VISTA fusion proteins may be usefultherapeutically for the treatment of conditions or disorders that wouldbenefit from modulation of the immune response. Moreover, theVISTA-fusion proteins of the invention can be used as immunogens toproduce anti-VISTA antibodies in a subject, to purify VISTA-bindingproteins, and in screening assays to identify molecules which inhibitthe interaction of VISTA with its natural binding partner.

Conjugates

The VISTA and VISTA conjugate, antibodies that bind the VISTA and VISTAconjugate and fragments thereof, may be conjugated to other moieties.Such conjugates are often used in the preparation of vaccines. The VISTAand VISTA conjugate polypeptide may be conjugated to a carbohydrate(e.g., mannose, fucose, glucose, GlcNAs, maltose), which is recognizedby the mannose receptor present on dendritic cells and macrophages. Theensuing binding, aggregation, and receptor-mediated endocytosis andphagocytosis functions provide enhanced innate and adaptive immunity SeeMahnke, et al. (2000) J. Cell Biol. 151: 673-84; Dong, et al. (1999) J.Immonol. 163: 5427-34.

Other moieties suitable for conjugation to elicit an immune responseincludes but not limited to Keyhole Limpit Hemocyannin (KLH), diphtheriatoxoid, cholera toxoid, Pseudomonas exoprotein A, and microbial outermembrane proteins (OMPS).

Polypeptide Isolation

The present invention also provides methods for isolation of the VISTAand VISTA conjugate polypeptides. For example, relevant cell lines ortumor samples may be obtained from a cancer patient. Afterhomogenization and solubilization in a detergent, the antigen ischromatographically purified. Size-exclusion or affinity chromatographymay be used for this, and may be used in conjunction with anti-VISTA andanti-VISTA-Ig conjugate antibodies. For example, anti-VISTA oranti-VISTA-Ig conjugate antibody may be immobilized on a solid support(e.g., coupled to resins, magnetic beads) for simple antigen adsorption,washing, and elution from the solid support. The eluted protein is thenstudied further for antigen presence, characterization, andidentification. See Walker (2002) Protein Protocols Handbook [2^(nd)Ed.] Humana Press and Culture (2003) [Ed.] Protein PurificationProtocols Humana Press.

The antigen isolated in this way may be used for preparing apharmaceutical using the conventional pharmaceutical excipient andcarrier substance. For example, in-vivo administration of the purifiedantigen in a physiological NaCl solution.

Additionally, the VISTA and VISTA conjugate polypeptides according tothe invention may serve as an antigen in the identification ofactivities as part of a high-throughput screening. High-throughputscreening methods are known to persons skilled in the art. Wells (2002)High Throughout Bioanalytical Sample Preparation Elsevier HealthSciences.

Polynucleotides Encoding VISTA and VISTA Conjugate

The present invention also provides nucleotides which encode VISTA andVISTA conjugates. The present invention also provides polynucleotidescomprising the nucleic acid sequences of SEQ ID NOs: 1 and 3 whichencode VISTA polypeptides. The present invention also provides forfragments, sequences hybridizable with, and sequences homologous to thepolynucleotide sequences described herein which are at least about 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%.

The invention also provides polynucleotides comprising at least oneVISTA and VISTA conjugate sequence encoding similar polypeptides withdifferent codon usage, altered sequences characterized by mutations,such as deletion, insertion or substitution of one or more nucleotides,either naturally occurring or man induced, either randomly or in atargeted fashion. The present invention also encompasses homologousnucleic acid sequences (e.g., which form a part of a polynucleotidesequence of the present invention), which include sequence regionsunique to the polynucleotides of the present invention.

The present invention also encompasses nucleic acids encoding homologuesof VISTA and VISTA conjugate polypeptides, such homologues can be atleast about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical homologous tothe amino acid sequences set forth herein, as may be determined usingBlastP software of the National Center of Biotechnology Information(NCBI) using default parameters. The present invention also encompassesfragments of the above described polynucleotides and polypeptides havingmutations, such as deletions, insertions or substitutions of one or morenucleic acids, either naturally occurring or man induced, eitherrandomly or in a targeted fashion.

Nucleic acid molecules may encode a VISTA and VISTA conjugate, or afunctional fragment of said nucleic acid molecule. A “functionalfragment” of said nucleic acid includes a fragment of the gene or cDNAencoding said VISTA and VISTA conjugate, which fragment is capable ofbeing expressed to produce a VISTA and VISTA conjugate capable ofeliciting an immune response (e.g., antibodies which selectively bindthe VISTA and VISTA conjugate) Thus, for example, fragments of the VISTAand VISTA conjugate according to the invention which correspond to aminoacid residues that contribute to the immunogenicity of the antigen andwhich fragments may serve to function as antigens to elicit an immuneresponse (e.g., humoral or cellular immune response.) This aspect of theinvention also includes differentially spliced isoforms andtranscriptional starts of the nucleic acids according to the invention.The nucleic acid molecules according to the invention also comprisefragments, derivatives and allelic variants of the nucleic acidmolecules described above that encodes a VISTA and VISTA conjugateaccording to the invention. Methods and materials for making nucleicacids encoding fragments of VISTA and VISTA conjugate are well known inthe art. See, e.g., Maniatis, et al. (2001) Molecular Cloning: ALaboratory Manual [3^(rd) Ed.] Cold Spring Harbor Laboratory Press.

A nucleic acid molecule encompassing all or a portion of SEQ ID NO: 1,3, or an ortholog or variant can be isolated by the polymerase chainreaction (PCR) using synthetic oligonucleotide primers designed basedupon the sequence of SEQ ID NO: 1, 2, 3, 4 or 5.

A nucleic acid molecule of the invention can be amplified using cDNA,mRNA or, alternatively, genomic DNA as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid molecule so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to VISTA (PD-L3) nucleotidesequences can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In an embodiment, an isolated VISTA encoding nucleic acid molecule ofthe invention comprises the nucleotide sequence shown in SEQ ID NO: 1 or3, or a fragment thereof. In another embodiment the nucleic acidmolecule of the invention comprises a nucleic acid molecule which is acomplement of the nucleotide sequence shown in SEQ ID NO: 1 or 3, or aportion of any of these nucleotide sequences. A nucleic acid moleculewhich is complementary to the nucleotide sequence shown in SEQ ID NO: 1or 3, is one which is sufficiently complementary to the nucleotidesequence shown in SEQ ID NO: 1 or 3 such that it can hybridize to thenucleotide sequence shown in SEQ ID NO: 1 or 3 respectively, therebyforming a stable duplex.

In another embodiment, an isolated nucleic acid molecule of the presentinvention comprises a nucleotide sequence which is at least about 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or99.5% identical to the entire length of the nucleotide sequence shown inSEQ ID NO: 1 or 3, or a portion of any of these nucleotide sequences.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO: 1 or 3, for example,a fragment which can be used as a probe or primer or a fragment whichencodes a portion of a VISTA polypeptide, e.g., a biologically activeportion of a VISTA-polypeptide. The nucleotide sequences determined fromthe cloning of the human PD-L2 gene allow for the generation of probesand primers designed for use in identifying and/or cloning other PD-L2family members, as well as VISTA homologues from other species. Theprobe/primer typically comprises substantially purified oligonucleotide.The oligonucleotide typically comprises a region of nucleotide sequencethat hybridizes under stringent conditions to at least about 12 or 15,preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55,60, 65, or 75 consecutive nucleotides of a sense sequence of SEQ ID NO:1 or 3; of an anti-sense sequence of SEQ ID NO: 1, 3, or a naturallyoccurring allelic variant or mutant of SEQ ID NO: 1 or 3.

In one embodiment, a nucleic acid molecule of the present inventioncomprises a nucleotide sequence which is greater than about 50-100,100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500,500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900,900-950, or more nucleotides in length and hybridizes under stringenthybridization conditions to a nucleic acid molecule of SEQ ID NO: 1 or3, or the complement thereof. In a further embodiment, a nucleic acidmolecule of the present invention comprises a nucleotide sequence whichis greater than about 880-900, 900-950, 950-1000, 1000-1050, 1050-1100,1100-1150, or more nucleotides in length and hybridizes under stringenthybridization conditions to a nucleic acid molecule of SEQ ID NO: 1 or3, or the complement thereof. In yet another embodiment, a nucleic acidmolecule of the present invention comprises a nucleotide sequence whichis greater than 50-100, 100-150, 150-200, 200-250, 250-300 or morenucleotides in length and hybridizes under stringent hybridizationconditions to a nucleic acid molecule comprising the coding region inSEQ ID NO: 1 or 3, or a complement thereof. In yet a further embodiment,a nucleic acid molecule of the present invention comprises a nucleotidesequence which is greater than about 50-100, 100-150, 150-200, 200-250,250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650,650-700, 700-750, 750-800, 850-900, 900-950, or more nucleotides inlength, includes at least about 15 (i.e., 15 contiguous) nucleotides ofthe sequence comprising the coding region of SEQ ID NO: 1 or 3, or acomplement thereof, and hybridizes under stringent conditions to anucleic acid molecule comprising the nucleotide sequence shown in SEQ IDNO: 1 or 3 a complement thereof.

Probes based on the VISTA nucleotide sequences can be used to detecttranscripts or genomic sequences encoding the same or homologouspolypeptides. In embodiments, the probe further comprises a label groupattached thereto, e.g., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a diagnostic test kit for identifying cells ortissue which misexpress a VISTA polypeptide, such as by measuring alevel of a VISTA-encoding nucleic acid in a sample of cells from asubject, e.g., detecting VISTA mRNA levels or determining whether agenomic VISTA gene has been mutated or deleted.

In addition to the VISTA nucleotide sequences of SEQ ID NO: 1 and 3, itwill be appreciated by those skilled in the art that DNA sequencepolymorphisms that lead to changes in the amino acid sequences of theVISTA polypeptides may exist within a population (e.g., the humanpopulation). Such genetic polymorphism in the VISTA genes may existamong individuals within a population due to natural allelic variation.As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules which include an open reading frame encoding a VISTApolypeptide, preferably a mammalian VISTA polypeptide, and can furtherinclude non-coding regulatory sequences, and introns.

Allelic variants of human or mouse VISTA include both functional andnon-functional VISTA polypeptides. Functional allelic variants arenaturally occurring amino acid sequence variants of the human or mouseVISTA polypeptide that maintain the ability to bind natural VISTAbinding partner(s) and/or modulate CD4+ and CD8+ T cell proliferationand cytokine production and lymphocyte activation. Functional allelicvariants will typically contain only conservative substitution of one ormore amino acids of SEQ ID NO: 2, 4 or 5, or substitution, deletion orinsertion of non-critical residues in non-critical regions of thepolypeptide.

Non-functional allelic variants are naturally occurring amino acidsequence variants of the human or mouse VISTA polypeptide that do nothave the ability to either bind natural VISTA binding partners, and/ormodulate any of the VISTA activities described herein. Non-functionalallelic variants will typically contain a non-conservative substitution,deletion, or insertion or premature truncation of the amino acidsequence of SEQ ID NO: 2, 4 or 5, or a substitution, insertion ordeletion in critical residues or critical regions of the polypeptide,e.g., in an IgV domain.

The present invention further provides non-human, non-mouse orthologs ofthe human or mouse VISTA polypeptide. Orthologs of the human or mouseVISTA polypeptide are polypeptides that are isolated from non-human,non-mouse organisms and possess the same binding activity and/orlymphocyte activation-modulating activity, and ability to modulate CD4+and CD8+ T cell proliferation and cytokine production as the human andmurine VISTA polypeptides disclosed herein. Orthologs of the human ormouse PD-L3 polypeptide can readily be identified as comprising an aminoacid sequence that is substantially identical to SEQ ID NO: 2, 4 or 5.

A mutant VISTA polypeptide may be assayed for the ability to bind toand/or modulate the activity of a natural VISTA binding partner, tomodulate intra- or intercellular signaling, modulate activation of Tlymphocytes, and/or modulate the immune response of an organism.

Isolated nucleic acid molecules encoding a VISTA or VISTA fusionproteins. Such nucleic acid molecules, comprising at least a firstnucleotide sequence encoding a VISTA or VISTA protein, polypeptide orpeptide operatively linked to a second nucleotide sequence encoding anon-VISTA protein, polypeptide or peptide, can be prepared by standardrecombinant DNA techniques.

Furthermore, identity refers broadly to the that functional and/orstructural equivalence that exists between the nucleic acid moleculesconcerned or the proteins coded by them. The nucleic acid molecules,which are homologous to the molecules described above and constitutederivatives of these molecules, are generally variations of thesemolecules, which constitute modifications, which execute the samebiological function. At the same time, the variations may occurnaturally, for example they may be sequences from other species, or theymay be mutants, wherein these mutants may have occurred in a naturalmanner or have been introduced by objective mutagenesis. The variationsmay also be synthetically manufactured sequences. The allelic variantsmay be both naturally occurring variants and also syntheticallymanufactured variants or variants produced by recombinant DNAtechniques. Nucleic acid molecules, which deviate from nucleic acidmolecules according to the invention due to degeneration of the geneticcode, constitute a special form of derivatives.

Included also within the scope of the invention is any nucleotidesequence that encodes the amino acid sequence of VISTA and VISTAconjugate thereof. Because the genetic code is degenerate, more than onecodon may be used to encode a particular amino acid. Using the geneticcode, one or more different nucleotides may be identified, each of whichwould be capable of encoding the amino acid. The probability that aparticular nucleotide will, in fact, constitute the actual codonencoding sequence may be estimated by considering abnormal base pairingrelationships and the frequency with which a particular codon isactually used (to encode a particular amino acid) in eukaryotic orprokaryotic cells expressing a VISTA and VISTA conjugate thereof. Such“codon usage rules” are disclosed by Lathe, et al. (1985) J. Molec.Biol. 183: 1-12.

Modified VISTA and VISTA Conjugate Polynucleotides

The nucleotides of the present invention may be modifiedpolynucleotides. Unmodified nucleotide are often less optimal in someapplications, e.g., prone to degradation by cellular nucleases. Chemicalmodifications to one or more of the subunits of oligonucleotide mayconfer improved properties, e.g., may render polynucleotides more stableto nucleases. Typical oligonucleotide modifications are well-known inthe art and may include one or more of: (i) alteration, e.g.,replacement, of one or both of the non-linking phosphate oxygens and/orof one or more of the linking phosphate oxygens in the phosphodiesterintersugar linkage; (ii) alteration, e.g., replacement, of a constituentof the ribose sugar, e.g., of the modification or replacement of the 2′hydroxyl on the ribose sugar; (iii) wholesale replacement of thephosphate moiety; (iv) modification or replacement of a naturallyoccurring base with a non-natural base; (v) replacement or modificationof the ribose-phosphate backbone, e.g. with peptide nucleic acid (PNA);(vi) modification of the 3′ end or 5′ end of the oligonucleotide; and(vii) modification of the sugar, e.g., six membered rings.Polynucleotides used in accordance with this invention may besynthesized by any number of means well-known in the art, or purchasedfrom a variety of commercial vendors (LC Sciences, Houston, Tex.;Promega, Madison, Wis.; Invitrogen, Carlsbad, Calif.).

Antisense

In addition to the nucleic acid molecules encoding VISTA polypeptidesdescribed above, another embodiment of the invention pertains toisolated nucleic acid molecules which are antisense thereto. An“antisense” nucleic acid comprises a nucleotide sequence which iscomplementary to a “sense” nucleic acid encoding a polypeptide, e.g.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence. Accordingly, an antisense nucleicacid can hydrogen bond to a sense nucleic acid. The antisense nucleicacid can be complementary to an entire VISTA coding strand, or to only aportion thereof. In one embodiment, an antisense nucleic acid moleculeis antisense to a “coding region” of the coding strand of a nucleotidesequence encoding a VISTA. The term “coding region” refers to the regionof the nucleotide sequence comprising codons which are translated intoamino acid residues. In another embodiment, the antisense nucleic acidmolecule is antisense to a “noncoding region” of the coding strand of anucleotide sequence encoding PD-L. The term “noncoding region” refers to5′ and 3′ sequences which flank the coding region that are nottranslated into amino acids (also referred to as 5′ and 3′ untranslatedregions). Given the coding strand sequences encoding human or mouseVISTA or VISTA disclosed herein, antisense nucleic acids of theinvention can be designed according to the rules of Watson and Crickbase pairing. The antisense nucleic acid molecule can be complementaryto the entire coding region of VISTA mRNA, but more preferably is anoligonucleotide which is antisense to only a portion of the coding ornoncoding region of VISTA mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of VISTA or VISTA mRNA. An antisenseoligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35,40, 45 or 50 nucleotides in length. An antisense nucleic acid moleculeof the invention can be constructed using chemical synthesis andenzymatic ligation reactions using procedures known in the art. Forexample, an antisense nucleic acid molecule (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, e.g., phosphorothioate derivatives and acridine substitutednucleotides can be used. Examples of modified nucleotides which can beused to generate the antisense nucleic acid include 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridin-e,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiour-acil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a VISTA orVISTA polypeptide to thereby inhibit expression of the polypeptide,e.g., by inhibiting transcription and/or translation. The hybridizationcan be by conventional nucleotide complementarity to form a stableduplex, or, for example, in the case of an antisense nucleic acidmolecule which binds to DNA duplexes, through specific interactions inthe major groove of the double helix. An example of a route ofadministration of antisense nucleic acid molecules of the inventioninclude direct injection at a tissue site. Alternatively, antisensenucleic acid molecules can be modified to target selected cells and thenadministered systemically. For example, for systemic administration,antisense molecules can be modified such that they specifically bind toreceptors or antigens expressed on a selected cell surface, e.g., bylinking the antisense nucleic acid molecules to peptides or antibodieswhich bind to cell surface receptors or antigens. The antisense nucleicacid molecules can also be delivered to cells using the vectorsdescribed herein. To achieve sufficient intracellular concentrations ofthe antisense molecules, vector constructs in which the antisensenucleic acid molecule is placed under the control of a strong pol II orpol III promoter are preferred.

The VISTA antisense nucleic acid molecule may be an α-anomeric nucleicacid molecule. An α-anomeric nucleic acid molecule forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other. Gaultier, et al.(1987) Nucleic Acids Res. 15: 6625-6641. The antisense nucleic acidmolecule can also comprise a 2′-O-methylribonucleotide (Inoue, et al.(1987) Nucleic Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analogue(Inoue, et al. (1987) FEBS Lett. 215: 327-330).

A VISTA antisense nucleic acid may be a ribozyme. Ribozymes arecatalytic RNA molecules with ribonuclease activity which are capable ofcleaving a single-stranded nucleic acid, such as an mRNA, to which theyhave a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes(described in Haseloff and Gerlach (1988) Nature 334:585-591)) can beused to catalytically cleave VISTA mRNA transcripts to thereby inhibittranslation of VISTA mRNA. A ribozyme having specificity for aVISTA-encoding nucleic acid can be designed based upon the nucleotidesequence of a VISTA cDNA disclosed herein (i.e., SEQ ID NO: 1 or 3). Forexample, a derivative of a Tetrahymena L-19 IVS RNA can be constructedin which the nucleotide sequence of the active site is complementary tothe nucleotide sequence to be cleaved in a VISTA-encoding mRNA. See,e.g., U.S. Pat. No. 4,987,071 and U.S. Pat. No. 5,116,742.Alternatively, VISTA mRNA can be used to select a catalytic RNA having aspecific ribonuclease activity from a pool of RNA molecules. See, e.g.,Bartel and Szostak (1993) Science 261:1411-1418.

Alternatively, VISTA gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of the VISTA(e.g., the VISTA promoter and/or enhancers; to form triple helicalstructures that prevent transcription of the PD-L3 gene in target cells.See generally, Helene (1991) Anticancer Drug Des. 6(6):569-84; Helene,et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992)Bioessays 14(12):807-15.

Peptide Nucleic Acid

In yet another embodiment, the VISTA nucleic acid molecules of thepresent invention can be modified at the base moiety, sugar moiety orphosphate backbone to improve, e.g., the stability, hybridization, orsolubility of the molecule. For example, the deoxyribose phosphatebackbone of the nucleic acid molecules can be modified to generatepeptide nucleic acids. See Hyrup and Nielsen (1996) Bioorg. Med. Chem.4(1): 5-23. As used herein, the terms “peptide nucleic acids” or “PNAs”refer to nucleic acid mimics, e.g, DNA mimics, in which the deoxyribosephosphate backbone is replaced by a pseudopeptide backbone and only thefour natural nucleobases are retained. The neutral backbone of PNAs hasbeen shown to allow for specific hybridization to DNA and RNA underconditions of low ionic strength. The synthesis of PNA oligomers can beperformed using standard solid phase peptide synthesis protocols asdescribed in Hyrup and Nielsen (1996) supra and Perry-O'Keefe et al.(1996) Proc Natl. Acad. Sci. USA 93:14670-675.

PNAs of VISTA nucleic acid molecules can be used in therapeutic anddiagnostic applications. For example, PNA scan be used as antisense orantigene agents for sequence-specific modulation of gene expression by,for example, inducing transcription or translation arrest or inhibitingreplication. PNAs of VISTA nucleic acid molecules can also be used inthe analysis of single base pair mutations in a gene (e.g., byPNA-directed PCR clamping); as ‘artificial restriction enzymes’ whenused in combination with other enzymes (e.g., 51 nucleases (Hyrup andNielsen (1996) supra)); or as probes or primers for DNA sequencing orhybridization (Hyrup and Nielsen (1996) supra; Perry-O'Keefe et al.(1996) supra).

PNAs of VISTA can be modified (e.g., to enhance their stability orcellular uptake), by attaching lipophilic or other helper groups to PNA,by the formation of PNA-DNA chimeras, or by the use of liposomes orother techniques of drug delivery known in the art. For example, PNA-DNAchimeras of VISTA nucleic acid molecules can be generated which maycombine the advantageous properties of PNA and DNA. Such chimeras allowDNA recognition enzymes (e.g., RNAse H and DNA polymerases), to interactwith the DNA portion while the PNA portion would provide high bindingaffinity and specificity. PNA-DNA chimeras can be linked using linkersof appropriate lengths selected in terms of base stacking, number ofbonds between the nucleobases, and orientation (Hyrup and Nielsen (1996)supra). The synthesis of PNA-DNA chimeras can be performed as describedin Hyrup and Nielsen (1996) supra and Finn P. J. et al. (1996) NucleicAcids Res. 24 (17):3357-63. For example, a DNA chain can be synthesizedon a solid support using standard phosphoramidite coupling chemistry andmodified nucleoside analogs, e.g.,5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can beused as a bridge between the PNA and the 5′ end of DNA (Mag, M. et al.(1989) Nucleic Acids Res. 17:5973-88). PNA monomers are then coupled ina stepwise manner to produce a chimeric molecule with a 5′ PNA segmentand a 3′ DNA segment (Finn P. J. et al. (1996) supra). Alternatively,chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNAsegment (Peterser, et al. (1975) Bioorganic Med. Chem. Lett.5:1119-11124).

Oligonucleotide

The oligonucleotide may include other appended groups such as peptides(e.g., for targeting host cell receptors in vivo), or agentsfacilitating transport across the cell membrane (See, e.g., Letsinger etal. (1989) Proc Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al.(1987) Proc Natl. Acad. Sci. USA 84:648-652; PCT Publication No. WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO89/10134). In addition, oligonucleotides can be modified withhybridization-triggered cleavage agents (See, e.g., Krol et al. (1988)Biotechniques 6:958-976) or intercalating agents (See, e.g., Zon (1988)Pharm. Res. 5:539-549). To this end, the oligonucleotide may beconjugated to another molecule (e.g., a peptide, hybridization triggeredcross-linking agent, transport agent, or hybridization-triggeredcleavage agent).

siRNA

Small interfering RNA (siRNA) are a class of double-stranded RNAmolecules usually about 20-25 nucleotides in length that bind to aspecific mRNA and direct it to mRNA degradation, thus suppressing thetranscrioption (e.g., expression) of the gene. See Hamilton & Baulcombe(1999) Science 286(5441): 950-2 and Elbashir, et al. (2001) Nature411(6836): 494-8. It is also possible to take advantage of ribozyme orRNA interference (siRNA) technology, which prevents a gene fromproducing a functional protein by destroying the messenger RNA. An siRNAmolecule may bind to VISTA mRNA transcribed from a VISTA DNA comprisingthe nucleic acid sequence of SEQ ID NO: 1 or 3. An siRNA molecule maybind to VISTA mRNA transcribed from a VISTA DNA encoding the amino acidsequence set forth in SEQ ID NO:2, 4 or 5.

An siRNA molecule which targets VISTA mRNA transcribed from a VISTA DNAmay comprise the nucleic acid sequence of SEQ ID NO: 1 or 3. An siRNAmolecule which targets VISTA mRNA transcribed from a VISTA DNA encodingthe amino acid sequence set forth in SEQ ID NO:2, 4 or 5. The siRNAmolecule that targets VISTA may comprise the nucleic acid sequence ofany one of SEQ ID NOs: 38-67. An siRNA molecule that targets either theORF or UTR region of VISTA may comprise the amino acid sequence of anyone of SEQ ID NO: 38-47. An siRNA molecule that targets the UTR regiononly of VISTA may comprise the amino acid sequence of any one of SEQ IDNO: 48-57. An siRNA molecule that targets the ORF region only of VISTAmay comprise the amino acid sequence of any one of SEQ ID NO: 58-67. AnsiRNA molecule that targets VISTA may consist of the nucleic acidsequence of any one of SEQ ID NOs: 38-67. An siRNA molecule that targetseither the ORF or UTR region of VISTA may consist of the amino acidsequence of any one of SEQ ID NO: 38-47. An siRNA molecule that targetsthe UTR region only of VISTA may consist the amino acid sequence of anyone of SEQ ID NO: 48-57. An siRNA molecule that targets the ORF regiononly of VISTA may consist the amino acid sequence of any one of SEQ IDNO: 58-67.

TABLE 1 siRNA for human VISTA Target region  SEQ  sRNA sequence of VISTAID NO GGGCACGATGTGACCTTCTACAAGA ORF 38 CAGATGCCAAATGACTTACATCTTA UTR3 39GAGATGGATTGTAAGAGCCAGTTTA UTR3 40 GGGCTTTGAGGAGAGGGTAAACATA UTR3 41CCTATCTCCTGACATTCACAGTTTA UTR3 42 CAGTTTAATAGAGACTTCCTGCCTT UTR3 43CAGGGAGAGGCTGAAGGAATGGAAT UTR3 44 GGAATGTGTTGAGAGGGATTCTGAA UTR3 45GAGAGGGATTCTGAATGATCAATAT UTR3 46 CACAGAGGGCAATAGAGGTTCTGAA UTR3 47CAGATGCCAAATGACTTACATCTTA UTR3 48 GAGATGGATTGTAAGAGCCAGTTTA UTR3 49GGTGAGTCCTCTGTGGAATTGTGAT UTR3 50 GGGCTTTGAGGAGAGGGTAAACATA UTR3 51CCTATCTCCTGACATTCACAGTTTA UTR3 52 CAGTTTAATAGAGACTTCCTGCCTT UTR3 53CAGGGAGAGGCTGAAGGAATGGAAT UTR3 54 GGAATGTGTTGAGAGGGATTCTGAA UTR3 55GAGAGGGATTCTGAATGATCAATAT UTR3 56 CACAGAGGGCAATAGAGGTTCTGAA UTR3 57ACAAAGGGCACGATGTGACCTTCTA ORF 58 GGGCACGATGTGACCTTCTACAAGA ORF 59GACCACCATGGCAACTTCTCCATCA ORF 60 CAGACAGGCAAAGATGCACCATCCA ORF 61GGCAAAGATGCACCATCCAACTGTG ORF 62 CCATCCAACTGTGTGGTGTACCCAT ORF 63GGATGGACAGCAACATTCAAGGGAT ORF 64 GACAGCAACATTCAAGGGATTGAAA ORF 65CCCTGTCCCTGACTCTCCAAACTTT ORF 66 CCTGACTCTCCAAACTTTGAGGTCA ORF 67Expression

Isolation and expression of the VISTA and VISTA conjugate of theinvention may be effected by well-established cloning procedures usingprobes or primers constructed based on the VISTA and VISTA conjugatenucleic acids sequences disclosed in the application. Related VISTA andVISTA conjugate sequences may also be identified from human or otherspecies genomic databases using the sequences disclosed herein and knowncomputer-based search technologies, e.g., BLAST sequence searching. Thepseudogenes disclosed herein may be used to identify functional allelesor related genes.

Expression vectors can then be used to infect or transfect host cellsfor the functional expression of these sequences. These genes andvectors can be made and expressed in vitro or in vivo. One of skill willrecognize that desired phenotypes for altering and controlling nucleicacid expression can be obtained by modulating the expression or activityof the genes and nucleic acids (e.g., promoters, enhancers) within thevectors of the invention. Any of the known methods described forincreasing or decreasing expression or activity can be used.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol 43:235-275), particular promoters of T cellreceptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example by the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3: 537-546).

The polynucleotide sequences provided herein may be generated accordingto any oligonucleotide synthesis method known in the art such asenzymatic synthesis or solid phase synthesis. Equipment and reagents forexecuting solid-phase synthesis are commercially available from, forexample, Applied Biosystems. Any other means for such synthesis may alsobe employed; the actual synthesis of the polynucleotides is well withinthe capabilities of one skilled in the art. See, e.g., Maniatis, et al.(2001) Molecular Cloning: A Laboratory Manual [3^(rd) Ed.] Cold SpringHarbor Laboratory Press; Swamy (2008) Laboratory Manual on BiotechnologyRastogi Publications; Herdewijn (2005) [Ed.] Methods in MolecularBiolog: Oligonucleotide Synthesis: Methods and Applications Volume 288Humana Press; and Rapley (2000) [Ed.] The Nucleic Acid ProtocolsHandbook Humana Press. Double-stranded DNA fragments may then beobtained either by synthesizing the complementary strand and annealingthe strands together under appropriate conditions, or by adding thecomplementary strand using DNA polymerase with an appropriate primersequence.

Techniques for the manipulation of nucleic acids, such as, for example,for generating mutations in sequences, subcloning, labeling probes,sequencing, hybridization are well described in the scientific andpatent literature. See, e.g., Sambrook, et al. (2001) (Eds.) MolecularCloning: A Laboratory Manual (3^(rd) Ed.) Cold Spring Harbor Laboratory;Ausubel, et al. (2011) Ed., Current Protocols in Molecular Biology, JohnWiley & Sons, Inc., New York; Tijssen (1993) [Ed.] Laboratory Techniquesin Biochemistry and Molecular Biology: Hybridization With Nucleic AcidProbes, Part I, Theory and Nucleic Acid Preparation, Elsevier, NY.

Hybridization and the strength of hybridization (e.g., the strength ofthe association between polynucleotides) is impacted by many factorswell known in the art including the degree of complementarity betweenthe polynucleotides, and the stringency of the conditions involved,which is affected by such conditions as the concentration of salts, thepresence of other components (e.g., the presence or absence ofpolyethylene glycol), the molarity of the hybridizing strands and theG+C content of the polynucleotide strands, all of which results in acharacteristic melting temperature (T_(m)) of the formed hybrid.Techniques of nucleic acid hybridization are disclosed by Sambrook, etal. (2001) (Eds.) Molecular Cloning: A Laboratory Manual [3^(rd) Ed.]Cold Spring Harbor Laboratory, and by Hayrnes, et al. (1985) in NUCLEICACID HYBRIDIZATION, A PRACTICAL APPROACH (IRL Press, DC). Hybridizationwash conditions may include wash solution of 0.2×SSC/0.1% SDS andincubation with rotation for 10 minutes at room temperature, (lowstringency wash), wash solution of prewarmed (42° C.) 0.2×SSC/0.1% SDSand incubation with rotation for 15 minutes at 42° C. (medium stringencywash) and wash solution of prewarmed (68° C.) 0.1×SSC/0.1% SDS andincubation with rotation for 15 minutes at 68° C. (high stringencywash). See Ausubel, et al. (2011) [Ed.] Current Protocols in MolecularBiology John Wiley & Sons, Inc.

Oligonucleotide primers may be used to amplify nucleic acids encoding aVISTA and VISTA conjugate. The nucleic acids described herein can alsobe cloned or measured quantitatively using amplification techniques.Amplification methods are also well known in the art, and include, e.g.,polymerase chain reaction (PCR) (Innis (1990) [Ed.] PCR Protocols, aGuide to Methods and Applications, Academic Press, NY.; Innis (1995)[Ed.] PCR Strategies, Academic Press, Inc., NY.); ligase chain reaction(LCR) (Wu (1989) Genomics 4: 560; Landegren (1988) Science 241: 1077;Barringer (1990) Gene 89: 117); transcription amplification (Kwoh (1989)PNAS 86: 1173); self-sustained sequence replication (Guatelli (1990)PNAS 87: 1874); Q Beta replicase amplification (Smith (1997) J. Clin.Microbiol. 35: 1477-91)); automated Q-beta replicase amplification assay(Burg (1996) Mol. Cell. Probes 10: 257-71); and other RNA polymerasemediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario). See,also, Berger (1987) Methods Enzymol. 152: 307-16; Sambrook, et al.(2001) (Eds.) Molecular Cloning: A Laboratory Manual (3^(rd) Ed.) ColdSpring Harbor Laboratory; Ausubel, et al. (2011) [Ed.] Current Protocolsin Molecular Biology, John Wiley & Sons, Inc., New York; Maniatis, etal. (2001) Molecular Cloning: A Laboratory Manual [3^(rd) Ed.] ColdSpring Harbor Laboratory Press; U.S. Pat. Nos. 4,683,195 and 4,683,202;Sooknanan (1995) Biotechnology 13: 563-64.

Paradigms to design degenerate primer pairs are well known in the art.For example, a COnsensus-DEgenerate Hybrid Oligonucleotide Primer(CODEHOP) strategy computer program is readily accessible and isdirectly linked from the BlockMaker multiple sequence alignment site forhybrid primer prediction beginning with a set of related proteinsequences, such as the VISTA and VISTA conjugate sequences providedherein. See, e.g., Rose (1998) Nucleic Acids Res. 26: 1628-35; Singh(1998) Biotechniques 24: 318-19.

Polymorphic variants, alleles, and interspecies homologs that aresubstantially identical to VISTA and VISTA conjugate disclosed hereinmay be isolated using the nucleic acid probes described above.Alternatively, expression libraries can be used to clone VISTA and VISTAconjugates and polymorphic variants, alleles, and interspecies homologsthereof, by detecting expressed homologs immunologically with antiseraor purified antibodies made against a VISTA and VISTA conjugate, whichalso recognize and selectively bind to the VISTA or VISTA conjugatehomolog.

Nucleic acids that encode VISTA and VISTA conjugate may be generated byamplification (e.g., PCR) of appropriate nucleic acid sequences usingappropriate (perfect or degenerate) primer pairs. The amplified nucleicacid can be genomic DNA from any cell or tissue or mRNA or cDNA derivedfrom VISTA or VISTA conjugate expressing cells. Methods for expressionof heterologous sequences in host cells are well known in the art. See,e.g., Maniatis, et al. (2001) Molecular Cloning: A Laboratory Manual[3^(rd) Ed.] Cold Spring Harbor Laboratory Press.

Fusion Proteins Comprising a VISTA and VISTA Conjugate

Hybrid protein-coding sequences comprising nucleic acids encoding VISTAand VISTA conjugate fused to a translocation sequences may beconstructed. Also provided are hybrid VISTA and VISTA conjugatecomprising the motifs and antigenic regions. These nucleic acidsequences may be operably linked to transcriptional or translationalcontrol elements, e.g., transcription and translation initiationsequences, promoters and enhancers, transcription and translationterminators, polyadenylation sequences, and other sequences useful fortranscribing DNA into RNA. In construction of recombinant expressioncassettes, vectors, and transgenics, a promoter fragment can be employedto direct expression of the desired nucleic acid in all desired cells ortissues.

Fusion proteins may comprise C-terminal or N-terminal translocationsequences. Further, fusion proteins can comprise additional elements,e.g., for protein detection, purification, or other applications.Detection and purification facilitating domains include, e.g., metalchelating peptides such as polyhistidine tracts, histidine-tryptophanmodules, or other domains that allow purification on immobilized metals;maltose binding protein; protein A domains that allow purification onimmobilized immunoglobulin; or the domain utilized in the FLAGSextension/affinity purification system (Immunex Corp, Seattle Wash.)

The inclusion of a cleavable linker sequences such as Factor Xa (see,e.g., Ottavi, (1998) Biochimie 80: 289-93), subtilisin proteaserecognition motif (see, e.g., Polyak (1997) Protein Eng. 10: 615-19);enterokinase (Invitrogen, San Diego, Calif.), between the translocationdomain (for efficient plasma membrane expression) and the rest of thenewly translated polypeptide may be useful to facilitate purification.For example, one construct can include a polypeptide encoding a nucleicacid sequence linked to six histidine residues followed by athioredoxin, an enterokinase cleavage site (see, e.g., Williams (1995)Biochemistry 34: 1787-97), and an C-terminal translocation domain. Thehistidine residues facilitate detection and purification while theenterokinase cleavage site provides a means for purifying the desiredprotein(s) from the remainder of the fusion protein. Technologypertaining to vectors encoding fusion proteins and application of fusionproteins are well described in the scientific and patent literature.See, e.g., Kroll (1993) DNA Cell. Biol. 12: 441-53.

Systems for Recombinant Expression of the VISTA and VISTA Conjugate

Expression vectors, either as individual expression vectors or aslibraries of expression vectors, comprising the ligand-binding regionencoding sequences may be introduced into a genome or into the cytoplasmor a nucleus of a cell and expressed by a variety of conventionaltechniques, well described in the scientific and patent literature. See,e.g., Sambrook, et al. (2001) [Eds.] Molecular Cloning: A LaboratoryManual (3^(rd) Ed.) Cold Spring Harbor Laboratory; Ausubel, et al.(2011) [Ed.] Current Protocols in Molecular Biology John Wiley & Sons,Inc.

The nucleic acids can be expressed in expression cassettes, vectors orviruses which are stably or transiently expressed in cells (e.g.,episomal expression systems). Selection markers can be incorporated intoexpression cassettes and vectors to confer a selectable phenotype ontransformed cells and sequences. For example, selection markers can codefor episomal maintenance and replication such that integration into thehost genome is not required. For example, the marker may encodeantibiotic resistance (e.g., chloramphenicol, kanamycin, G418,bleomycin, hygromycin) or herbicide resistance (e.g., chlorosulfurone orBasta) to permit selection of those cells transformed with the desiredDNA sequences. See, e.g., Ausubel, et al. (2011) [Ed.] Current Protocolsin Molecular Biology John Wiley & Sons, Inc.; and Walker & Papley (2009)Molecular Biology and Biotechnology [5^(th) Ed.] Royal Society ofChemistry. Because selectable marker genes conferring resistance tosubstrates like neomycin or hygromycin can only be utilized in tissueculture, chemoresistance genes are also used as selectable markers invitro and in vivo.

To enable cellular expression of the polynucleotides of the presentinvention, a nucleic acid construct according to the present inventionmay be used, which includes at least a coding region of one of the abovenucleic acid sequences, and further includes at least one cis actingregulatory element. Preferably, the promoter utilized by the nucleicacid construct of the present invention is active in the specific cellpopulation transformed. Examples of cell type-specific and/ortissue-specific promoters are well-known in the art. See Bernardi (2003)[Ed.] Gene Transfer and Expression in Mammalian Cells Volume 38 ElsevierScience B.V. The nucleic acid construct of the present invention canfurther include an enhancer, which can be adjacent or distant to thepromoter sequence and can function in up regulating the transcriptiontherefrom.

The nucleic acid construct of the present invention preferably furtherincludes an appropriate selectable marker and/or an origin ofreplication. Preferably, the nucleic acid construct utilized is ashuttle vector, which can propagate both in E. coli (wherein theconstruct comprises an appropriate selectable marker and origin ofreplication) and be compatible for propagation in cells, or integrationin a gene and a tissue of choice. The construct according to the presentinvention can be, for example, a plasmid, a bacmid, a phagemid, acosmid, a phage, a virus or an artificial chromosome.

Examples of suitable constructs include, but are not limited to, pcDNA3,pcDNA3.1 (+/−), pGL3, PzeoSV2 (+/−), pDisplay, pEF/myc/cyto,pCMV/myc/cyto each of which is commercially available from InvitrogenCo. (Carlsbad, Calif.) Examples of retroviral vector and packagingsystems are those sold by Clontech (San Diego, Calif.), includingRetro-X vectors pLNCX and pLXSN, which permit cloning into multiplecloning sites and the transgene is transcribed from CMV promoter.Vectors derived from Mo-MuLV are also included such as pBabe, where thetransgene will be transcribed from the 5′ LTR promoter.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, that is operatively-linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably-linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerthat allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell).

The term “regulatory sequence” is intended to includes promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel (1990) Gene Expression Technology Methods in Enzymology 185,Academic Press, San Diego, Calif. Regulatory sequences include thosethat direct constitutive expression of a nucleotide sequence in manytypes of host cell and those that direct expression of the nucleotidesequence only in certain host cells (e.g., tissue-specific regulatorysequences). It will be appreciated by those skilled in the art that thedesign of the expression vector can depend on such factors as the choiceof the host cell to be transformed, the level of expression of proteindesired. The expression vectors of the invention can be introduced intohost cells to thereby produce proteins or peptides, including fusionproteins or peptides, encoded by nucleic acids as described herein.

The recombinant expression vectors of the invention may be designed forproduction of variant proteins in prokaryotic or eukaryotic cells. Forexample, proteins of the invention can be expressed in bacterial cellssuch as Escherichia coli, insect cells (e.g., using baculovirusexpression vectors), yeast cells, or mammalian cells. Suitable hostcells are discussed further in Goeddel (1990) Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example using T7 promoter regulatory sequencesand T7 polymerase.

Expression of proteins in prokaryotes is most often carried out inEscherichia coli with vectors containing constitutive or induciblepromoters directing the expression of either fusion or non-fusionproteins. Fusion vectors add a number of amino acids to a proteinencoded therein, to the amino or C terminus of the recombinant protein.Such fusion vectors typically serve three purposes: (i) to increaseexpression of recombinant protein; (ii) to increase the solubility ofthe recombinant protein; and (iii) to aid in the purification of therecombinant protein by acting as a ligand in affinity purification.Often, in fusion expression vectors, a proteolytic cleavage site isintroduced at the junction of the fusion moiety and the recombinantprotein to enable separation of the recombinant protein from the fusionmoiety subsequent to purification of the fusion protein. Such enzymes,and their cognate recognition sequences, include Factor Xa, thrombin,PreScission, TEV and enterokinase. Typical fusion expression vectorsinclude pGEX (Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose Ebinding protein, or protein A, respectively, to the target recombinantprotein.

The recombinant mammalian expression vector is capable of directingexpression of the nucleic acid may be in a particular cell type (e.g.,tissue-specific regulatory elements are used to express the nucleicacid). Tissue-specific regulatory elements are known in the art. Forefficient production of the protein, it is preferable to place thenucleotide sequences encoding the protein of the invention under thecontrol of expression control sequences optimized for expression in adesired host. For example, the sequences may include optimizedtranscriptional and/or translational regulatory sequences (e.g., alteredKozak sequences).

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacterium with an impaired capacity toproteolytically cleave the recombinant protein. See, e.g., Gottesman(1990) Gene Expression Technology Methods in Enzymology Academic Press,San Diego, Calif. 185: 119-128. Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli. See, e.g., Wada, et al. (1992) Nucl.Acids Res. 20: 2111-2118. Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques.Another strategy to solve codon bias is by using BL21-codon plusbacterial strains (Invitrogen) or Rosetta bacterial strain (Novagen),these strains contain extra copies of rare E. coli tRNA genes.

The expression vector encoding for the protein of the invention may be ayeast expression vector. Examples of vectors for expression in yeastSaccharomyces cerevisiae include pYepSec1 (Baldari, et al. (1987) EMBOJ. 6: 229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30: 933-943),pJRY88 (Schultz, et al. (1987) Gene 54: 113-123), pYES2 (InvitrogenCorporation, San Diego, Calif.), and picZ (Invitrogen Corp, San Diego,Calif.)

Alternatively, polypeptides of the present invention can be produced ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., SF9cells) include the pAc series (Smith, et al. (1983) Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39). In yet another embodiment, a nucleic acid of the invention isexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed (1987)Nature 329: 840) and pMT2PC (Kaufman, et al. (1987) EMBO J. 6: 187-195),pIRESpuro (Clontech), pUB6 (Invitrogen), pCEP4 (Invitrogen) pREP4(Invitrogen), pcDNA3 (Invitrogen). When used in mammalian cells, theexpression vector's control functions are often provided by viralregulatory elements. For example, commonly used promoters are derivedfrom polyoma, adenovirus 2, cytomegalovirus, Rous Sarcoma Virus, andsimian virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see, e.g., Sambrook, et al. (2001)(Eds.) Molecular Cloning: A Laboratory Manual (3^(rd) Ed.) Cold SpringHarbor Laboratory.

A host cell can be any prokaryotic or eukaryotic cell. For example,protein of the invention can be produced in bacterial cells such as E.coli, insect cells, yeast, plant or mammalian cells (e.g., Chinesehamster ovary cells (CHO), COS, HEK293 cells). Other suitable host cellsare known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (2001) [Eds.]Molecular Cloning: A Laboratory Manual (3^(rd) Ed.) Cold Spring HarborLaboratory and other laboratory manuals.

Any of the well-known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA or other foreign genetic material into a host cell. See,e.g., Sambrook, et al. (2001) (Eds.) Molecular Cloning: A LaboratoryManual (3^(rd) Ed.) Cold Spring Harbor Laboratory and Walker & Papley(2009) Molecular Biology and Biotechnology [5^(th) Ed.] Royal Society ofChemistry. It is only necessary that the particular genetic engineeringprocedure used be capable of successfully introducing at lest onenucleic acid molecule into the host cell capable of expressing the VISTAand VISTA conjugate, fragment, or variant of interest.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest. Variousselectable markers include those that confer resistance to drugs, suchas G418, hygromycin, puromycin, blasticidin and methotrexate. Nucleicacids encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding protein of the invention or can beintroduced on a separate vector. Cells stably transfected with theintroduced nucleic acid can be identified by drug selection (e.g., cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) protein of theinvention. Accordingly, the invention further provides methods forproducing proteins of the invention using the host cells of theinvention. In one embodiment, the method comprises culturing the hostcell of the present invention (into which a recombinant expressionvector encoding protein of the invention has been introduced) in asuitable medium such that the protein of the invention is produced. Inanother embodiment, the method further comprises isolating protein ofthe invention from the medium or the host cell.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofthe receptor, fragment, or variant of interest, which is then recoveredfrom the culture using standard techniques. Examples of such techniquesare well known in the art. See, e.g., WO 00/06593.

Antibodies which Bind VISTA or VISTA Conjugates

The present invention also provides antibodies which selectively bindthe VISTA and VISTA conjugate including but not limited monoclonal andhumanized monoclonal antibodies. The antibodies which selectively bindthe VISTA and VISTA conjugate may be admixed in compositions withpharmaceutical carriers and additional antibodies (e.g., anti-PD-L1,PD-L2 or CTLA-4 antibodies).

An isolated VISTA polypeptide, or a portion or fragment thereof, can beused as an immunogen to generate antibodies that bind VISTA usingstandard techniques for polyclonal and monoclonal antibody preparation.A full-length VISTA polypeptide can be used or, alternatively, theinvention provides antigenic peptide fragments of VISTA for use asimmunogens. In one embodiment, an antigenic peptide of VISTA comprisesat least 8 amino acid residues of the amino acid sequence shown in SEQID NO: 2, 4 or 5 and encompasses an epitope of VISTA such that anantibody raised against the peptide forms a specific immune complex withthe VISTA polypeptide. Preferably, the antigenic peptide comprises atleast 10 amino acid residues, more preferably at least 15 amino acidresidues, even more preferably at least 20 amino acid residues, and mostpreferably at least 30 amino acid residues. Preferred epitopesencompassed by the antigenic peptide are regions of VISTA that arelocated in the extracellular domain of the polypeptide, e.g.,hydrophilic regions, as well as regions with high antigenicity.

A VISTA immunogen typically is used to prepare antibodies by immunizinga suitable subject (e.g., rabbit, goat, mouse, or other mammal) with theimmunogen. An appropriate immunogenic preparation can contain, forexample, recombinantly expressed VISTA polypeptide or a chemicallysynthesized VISTA polypeptide. The preparation can further include anadjuvant, such as Freund's complete or incomplete adjuvant, or similarimmunostimulatory agent. Immunization of a suitable subject with animmunogenic VISTA preparation induces a polyclonal anti-VISTA antibodyresponse.

Antibodies may comprise of two identical light polypeptide chains ofmolecular weight approximately 23,000 daltons (“light chain”), and twoidentical heavy chains of molecular weight 53,000-70,000 (“heavychain”). See Edelman (1971) Ann. NY. Acad. Sci. 190: 5. The four chainsare joined by disulfide bonds in a “Y” configuration wherein the lightchains bracket the heavy chains starting at the mouth of the “Y”configuration. The “branch” portion of the “Y” configuration isdesignated the F_(ab) region; the stem portion of the “Y” configurationis designated the F_(c) region. The amino acid sequence orientation runsfrom the N-terminal end at the top of the “Y” configuration to theC-terminal end at the bottom of each chain. The N-terminal end possessesthe variable region having specificity for the antigen that elicited it,and is about 100 amino acids in length, there being slight variationsbetween light and heavy chain and from antibody to antibody.

The variable region is linked in each chain to a constant region thatextends the remaining length of the chain and that within a particularclass of antibody does not vary with the specificity of the antibody(i.e., the antigen eliciting it). There are five known major classes ofconstant regions that determine the class of the immunoglobulin molecule(e.g., IgG, IgM, IgA, IgD, and IgE corresponding to γ, μ, α, δ, and εheavy chain constant regions). The constant region or class determinessubsequent effector function of the antibody, including activation ofcomplement (Kabat (1976) Structural Concepts in Immunology andImmunochemistry [2^(nd) Ed.] pages 413-436; Holt, Rinehart, Winston) andother cellular responses (Andrews, et al. (1980) Clinical Immunobiology1-18; Kohl, et al. (1983) Immunology 48: 187) while the variable regiondetermines the antigen with which it will react. Light chains areclassified as either κ (kappa) or λ (lambda). Each heavy chain class maybe prepared with either kappa or lambda light chain. The light and heavychains are covalently bonded to each other, and the “tail” portions ofthe two heavy chains are bonded to each other by covalent disulfidelinkages when the immunoglobulins are generated either by hybridomas orby B cells.

Specific binding to an antibody under such conditions may require anantibody that is selected for its specificity for a particular protein.For example, polyclonal antibodies raised to seminal basic protein fromspecific species such as rat, mouse, or human can be selected to obtainonly those polyclonal antibodies that are specifically immunoreactivewith seminal basic protein and not with other proteins, except forpolymorphic variants and alleles of seminal basic protein. Thisselection may be achieved by subtracting out antibodies that cross-reactwith seminal basic protein molecules from other species. A variety ofimmunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select antibodies specificallyimmunoreactive with a protein. See, e.g., Harlow & Lane (1998) USINGANTIBODIES: A LABORATORY MANUAL Cold Spring Harbor Laboratory, for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity. Typically a specific or selectivereaction will be at least twice background signal or noise and moretypically more than about 10 to 100 times background.

Antibodies may be screened to identify those that bind to specificepitopes of VISTA, e.g. in the IgV domain or other specific domainsand/or to select antibodies possessing high affinity and avidity toVISTA protein. In addition these antibodies are screened to identifythose of which modulate specific functions and effects of VISTA onimmunity and immune cells in vitro and in vivo. For example assays canbe conducted to ascertain the modulatory effect, if any, of a particularanti-VISTA antibody on immune functions negatively regulated by VISTAincluding cytokine production by CD4+ or CD8+ T cells, CD28costimulation, CD4+ T cell proliferation, and the proliferation of naïveand memory CD4+ T cells, et al. In an embodiment assays are conducted toidentify potential therapeutic anti-VISTA antibodies which in vitro,when the presence of VISTA-Ig enhance the suppression by VISTA-Ig asthese anti-VISTA antibodies behave oppositely in vivo, i.e., they areimmunosuppressive. The invention encompasses anti-VISTA antibodies anduse thereof that specifically bind to the 136 amino acid extracellulardomain, e.g., to amino acids 1-50, 50-100, 100-136, antibodies thatspecifically bind the IgV, antibodies that specifically bind the stalkregion, antibodies that specifically bind the transmembrane region andantibodies that specifically bind the cytoplasmic region of VISTA. Thesespecific regions are identified in the application.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol 43:235-275), particular promoters of T cellreceptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example by the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3: 537-546).

Polyclonal Antibody

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen.Polyclonal antibodies which selectively bind the VISTA and VISTAconjugate may be made by methods well-known in the art. See, e.g.,Howard & Kaser (2007) Making and Using Antibodies: A Practical HandbookCRC Press.

Monoclonal Antibody

A monoclonal antibody contains a substantially homogeneous population ofantibodies specific to antigens, which population contains substantiallysimilar epitope binding sites. Monoclonal antibodies may be obtained bymethods known to those skilled in the art. See, e.g. Kohler and Milstein(1975) Nature 256: 495-497; U.S. Pat. No. 4,376,110; Ausubel, et al.[Eds.] (2011) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene PublishingAssoc. and Wiley Interscience, NY.; and Harlow & Lane (1998) USINGANTIBODIES: A LABORATORY MANUAL Cold Spring Harbor Laboratory; Colligan,et al. (2005) [Eds.] Current Protocols in Immunology Greene PublishingAssoc. and Wiley Interscience, NY. Such antibodies may be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, GILD and any subclassthereof. A hybridoma producing an antibody of the present invention maybe cultivated in vitro, in situ, or in vivo.

Chimeric Antibody

Chimeric antibodies are molecules different portions of which arederived from different animal species, such as those having variableregion derived from a murine antibody and a human immunoglobulinconstant region, which are primarily used to reduce immunogenicity inapplication and to increase yields in production, for example, wheremurine monoclonal antibodies have higher yields from hybridomas buthigher immunogenicity in humans, such that human murine chimericmonoclonal antibodies are used. Chimeric antibodies and methods fortheir production are known in the art. See Cabilly, et al. (1984) Proc.Natl. Acad. Sci. USA 81: 3273-3277; Morrison, et al. (1994) Proc. Natl.Acad. Sci. USA 81: 6851-6855, Boulianne, et al. (1984) Nature 312:643-646; Neuberger, et al. (1985) Nature 314: 268-270; European PatentApplication 173494 (1986); WO 86/01533 (1986); European PatentApplication 184187 (1986); European Patent Application 73494 (1986);Sahagan, et al. (1986) J. Immunol. 137: 1066-1074; Liu, et al. (1987)Proc. Natl. Acad. Sci. USA 84: 3439-3443; Sun, et al. (1987) Proc. Natl.Acad. Sci. USA 84: 214-218; Better, et al. (1988) Science 240:1041-1043; and Harlow & Lane (1998) USING ANTIBODIES: A LABORATORYMANUAL Cold Spring Harbor Laboratory; U.S. Pat. No. 5,624,659.

Humanized Antibody

Humanized antibodies are engineered to contain even more human-likeimmunoglobulin domains, and incorporate only thecomplementarity-determining regions of the animal-derived antibody. Thismay be accomplished by examining the sequence of the hyper-variableloops of the variable regions of the monoclonal antibody, and fittingthem to the structure of the human antibody chains. See, e.g., U.S. Pat.No. 6,187,287. Likewise, other methods of producing humanized antibodiesare now well known in the art. See, e.g., U.S. Pat. Nos. 5,225,539;5,530,101; 5,585,089; 5,693,762; 6,054,297; 6,180,370; 6,407,213;6,548,640; 6,632,927; and 6,639,055; Jones, et al. (1986) Nature 321:522-525; Reichmann, et al. (1988) Nature 332: 323-327; Verhoeyen, et al.(1988) Science 239: 1534-36; and Zhiqiang An (2009) [Ed.] TherapeuticMonoclonal Antibodies: From Bench to Clinic John Wiley & Sons, Inc.

Antibody Fragments

In addition to entire immunoglobulins (or their recombinantcounterparts), immunoglobulin fragments comprising the epitope bindingsite (e.g., Fab′, F(ab′)₂, or other fragments) may be synthesized.“Fragment,” or minimal immunoglobulins may be designed utilizingrecombinant immunoglobulin techniques. For instance “Fv” immunoglobulinsfor use in the present invention may be produced by synthesizing a fusedvariable light chain region and a variable heavy chain region.Combinations of antibodies are also of interest, e.g. diabodies, whichcomprise two distinct Fv specificities. Antigen-binding fragments ofimmunoglobulins include but are not limited to SMIPs (small moleculeimmunopharmaceuticals), camelbodies, nanobodies, and IgNAR.

Anti-Idiotypic Antibody

An anti-idiotypic (anti-Id) antibody is an antibody which recognizesunique determinants generally associated with the antigen-binding siteof an antibody. An Id antibody may be prepared by immunizing an animalof the same species and genetic type (e.g., mouse strain) as the sourceof the antibody with the antibody to which an anti-Id is being prepared.The immunized animal will recognize and respond to the idiotypicdeterminants of the immunizing antibody by producing an antibody tothese idiotypic determinants (the anti-ld antibody). See e.g., U.S. Pat.No. 4,699,880. The anti-Id antibody may also be used as an “immunogen”to induce an immune response in yet another animal, producing aso-called anti-anti-Id antibody. The anti-anti-Id may be epitopicallyidentical to the original antibody which induced the anti-Id. Thus, byusing antibodies to the idiotypic determinants of an antibody it ispossible to identify other clones expressing antibodies of identicalspecificity.

Engineered and Modified Antibodies

An antibody of the invention further may be prepared using an antibodyhaving one or more of the VH and/or VL sequences derived from anantibody starting material to engineer a modified antibody, whichmodified antibody may have altered properties from the startingantibody. An antibody may be engineered by modifying one or moreresidues within one or both variable regions (i.e., VH and/or VL), forexample within one or more CDR regions and/or within one or moreframework regions. Additionally or alternatively, an antibody may beengineered by modifying residues within the constant region(s), forexample to alter the effector function(s) of the antibody.

One type of variable region engineering that may be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties. See, e.g., Riechmann, et al. (1998) Nature 332:323-327; Jones, et al. (1986) Nature 321: 522-525; Queen, et al. (1989)Proc. Natl. Acad. U.S.A. 86: 10029-10033; U.S. Pat. Nos. 5,225,539;5,530,101; 5,585,089; 5,693,762; and 6,180,370.

Suitable framework sequences may be obtained from public DNA databasesor published references that include germline antibody gene sequences.For example, germline DNA sequences for human heavy and light chainvariable region genes may be found in the “VBase” human germlinesequence database (available on the Internet), as well as in Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest [5thEd.]U.S. Department of Health and Human Services, NIH Publication No.91-3242; Tomlinson, et al. (1992) “The Repertoire of Human Germline VHSequences Reveals about Fifty Groups of VH Segments with DifferentHypervariable Loops” J. Mol. Biol. 227: 776-798; and Cox, et al. (1994)Eur. J Immunol 24: 827-836.

Another type of variable region modification is to mutate amino acidresidues within the VH and/or VL CDR 1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest. Site-directed mutagenesis or PCR-mediatedmutagenesis may be performed to introduce the mutation(s) and the effecton antibody binding, or other functional property of interest, may beevaluated in appropriate in vitro or in vivo assays. Preferablyconservative modifications (as discussed herein) may be introduced. Themutations may be amino acid substitutions, additions or deletions, butare preferably substitutions. Moreover, typically no more than one, two,three, four or five residues within a CDR region are altered.

Engineered antibodies of the invention include those in whichmodifications have been made to framework residues within VH and/or VL,e.g. to improve the properties of the antibody. Typically such frameworkmodifications are made to decrease the immunogenicity of the antibody.For example, one approach is to “backmutate” one or more frameworkresidues to the corresponding germline sequence. More specifically, anantibody that has undergone somatic mutation may contain frameworkresidues that differ from the germline sequence from which the antibodyis derived. Such residues may be identified by comparing the antibodyframework sequences to the germline sequences from which the antibody isderived.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the invention may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of the invention may bechemically modified (e.g., one or more chemical moieties may be attachedto the antibody) or be modified to alter its glycosylation, again toalter one or more functional properties of the antibody. Suchembodiments are described further below. The numbering of residues inthe Fc region is that of the EU index of Kabat.

The hinge region of CH1 may be modified such that the number of cysteineresidues in the hinge region is altered, e.g., increased or decreased.See U.S. Pat. No. 5,677,425. The number of cysteine residues in thehinge region of CH1 may be altered to, for example, facilitate assemblyof the light and heavy chains or to increase or decrease the stabilityof the antibody.

The Fc hinge region of an antibody may be mutated to decrease thebiological half life of the antibody. More specifically, one or moreamino acid mutations may be introduced into the CH2-CH3 domain interfaceregion of the Fc-hinge fragment such that the antibody has impairedStaphylococcyl protein A (SpA) binding relative to native Fc-hingedomain SpA binding. See, e.g., U.S. Pat. No. 6,165,745.

The antibody may be modified to increase its biological half life.Various approaches are possible. For example, one or more of thefollowing mutations may be introduced: T252L, T254S, T256F. See U.S.Pat. No. 6,277,375. Alternatively, to increase the biological half life,the antibody may be altered within the CH1 or CL region to contain asalvage receptor binding epitope taken from two loops of a CH2 domain ofan Fc region of an IgG. See U.S. Pat. Nos. 5,869,046 and 6,121,022.

The Fc region may be altered by replacing at least one amino acidresidue with a different amino acid residue to alter the effectorfunction(s) of the antibody. For example, one or more amino acidsselected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and322 may be replaced with a different amino acid residue such that theantibody has an altered affinity for an effector ligand but retains theantigen-binding ability of the parent antibody. The effector ligand towhich affinity may be altered may be, for example, an Fc receptor or theC1 component of complement. See U.S. Pat. Nos. 5,624,821 and 5,648,260.

The glycosylation of an antibody may be modified. For example, anaglycoslated antibody may be made (i.e., the antibody lacksglycosylation). Glycosylation may be altered to, for example, increasethe affinity of the antibody for antigen. Such carbohydratemodifications may be accomplished by, for example, altering one or moresites of glycosylation within the antibody sequence. For example, one ormore amino acid substitutions may be made that result in elimination ofone or more variable region framework glycosylation sites to therebyeliminate glycosylation at that site. Such aglycosylation may increasethe affinity of the antibody for antigen. See, e.g., U.S. Pat. Nos.5,714,350 and 6,350,861.

Additionally or alternatively, an antibody may be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications may be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and may be used as host cells in which to expressrecombinant antibodies of the invention to thereby produce an antibodywith altered glycosylation. See U.S. Patent Application Publication No.2004/0110704 and Yamane-Ohnuki, et al. (2004) Biotechnol Bioeng. 87:614-22; EP 1,176,195; WO 2003/035835; Shields, et al. (2002) J. Biol.Chem. 277: 26733-26740; WO 99/54342; Umana, et al. (1999) Nat. Biotech.17: 176-180; and Tarentino, et al. (1975) Biochem. 14: 5516-23.

An antibody may be Pegylated to, for example, increase the biological(e.g., serum) half life of the antibody. To pegylate an antibody, theantibody, or fragment thereof, typically is reacted with polyethyleneglycol (PEG), such as a reactive ester or aldehyde derivative of PEG,under conditions in which one or more PEG groups become attached to theantibody or antibody fragment. Preferably, the pegylation is carried outvia an acylation reaction or an alkylation reaction with a reactive PEGmolecule (or an analogous reactive water-soluble polymer).

The invention also provides variants and equivalents that aresubstantially homologous to the antibodies, antibody fragments,diabodies, SMIPs, camelbodies, nanobodies, IgNAR, polypeptides, variableregions and CDRs set forth herein. These may contain, e.g., conservativesubstitution mutations, (i.e., the substitution of one or more aminoacids by similar amino acids). For example, conservative substitutionrefers to the substitution of an amino acid with another within the samegeneral class, e.g., one acidic amino acid with another acidic aminoacid, one basic amino acid with another basic amino acid, or one neutralamino acid by another neutral amino acid.

Antibody Conjugates

Further, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Methods of Engineering Antibodies

Antibodies having VH and VL sequences disclosed herein may be used tocreate new variant antibodies by modifying the VH and/or VL sequences,or the constant region(s) attached thereto. Thus, the structuralfeatures of an variant antibody of the invention, are used to createstructurally related variant antibodies that retain at least onefunctional property of the antibodies of the invention, such as bindingto VISTA and VISTA conjugate. For example, one or more CDR regions ofone Anti-VISTA variant antibody or anti-VISTA conjugate variantantibody, or mutations thereof, may be combined recombinantly with knownframework regions and/or other CDRs to create additional,recombinantly-engineered, anti-VISTA or anti-VISTA conjugate antibodies(e.g., antibodies which bind the VISTA and VISTA conjugate) of theinvention, as discussed herein. The starting material for theengineering method may be one or more of the VH and/or VK sequencesprovided herein, or one or more CDR regions thereof. To create theengineered antibody, it is not necessary to actually prepare (i.e.,express as a protein) an antibody having one or more of the VH and/or VKsequences provided herein, or one or more CDR regions thereof. Rather,the information contained in the sequence(s) is used as the startingmaterial to create a “second generation” sequence(s) derived from theoriginal sequence(s) and then the “second generation” sequence(s) isprepared and expressed as a protein. Standard molecular biologytechniques may be used to prepare and express altered antibody sequence.

The antibody encoded by the altered antibody sequence(s) may retain one,some or all of the functional properties of the anti-VISTA or anti-VISTAconjugate antibodies produced by methods and with sequences providedherein, which functional properties include binding to variant VISTA orvariant VISTA conjugate with a specific KD level or less and/ormodulating immune cell activity, and/or selectively binding to desiredtarget cells such as, for example, colorectal carcinoma, lung cancer,prostate cancer, pancreas cancer, ovarian cancer, gastric cancer, andliver cancer. The functional properties of the altered antibodies may beassessed using standard assays available in the art and/or describedherein.

Mutations may be introduced randomly or selectively along all or part ofan anti-VISTA or anti-VISTA conjugate antibody coding sequence and theresulting modified anti-VISTA or anti-VISTA conjugate antibodies may bescreened for binding activity and/or other desired functionalproperties. See WO 2011/120013.

Nucleic Acids Encoding Antibodies that Selectively Bind VISTA or VISTAConjugate

Another embodiment of the invention pertains to nucleic acid moleculesthat encode the antibodies of the invention which bind the VISTA andVISTA conjugate. The nucleic acids may be present in whole cells, in acell lysate, or in a partially purified or substantially pure form. Anucleic acid may be isolated by purification away from other cellularcomponents or other contaminants (e.g., other cellular nucleic acids orproteins) by standard techniques, including alkaline/SDS treatment, CsClbanding, column chromatography, agarose gel electrophoresis and otherswell known in the art. See Ausubel, et al. (2011) Current Protocols inMolecular Biology John Wiley & Sons, Inc. A nucleic acid of theinvention may be, for example, DNA or RNA and may or may not containintronic sequences. The nucleic acid may be a cDNA molecule.

Nucleic acids of the invention may be obtained using standard molecularbiology techniques. For antibodies expressed by hybridomas (e.g.,hybridomas prepared from transgenic mice carrying human immunoglobulingenes as described further below), cDNAs encoding the light and heavychains of the antibody made by the hybridoma may be obtained by standardPCR amplification or cDNA cloning techniques. For antibodies obtainedfrom an immunoglobulin gene library (e.g., using phage displaytechniques), nucleic acid encoding the antibody may be recovered fromthe library.

Specifically, degenerate codon substitutions may be achieved bygenerating, e.g., sequences in which the third position of one or moreselected codons is substituted with mixed-base and/or deoxyinosineresidues. Batzer, et al. (1991) Nucleic Acid Res. 19: 5081; Ohtsuka, etal. (1985) J. Biol. Chem. 260: 2605-08; Rossolini, et al. (1994) Mol.Cell. Probes 8: 91-98.

Once DNA fragments encoding VH and VL segments are obtained, these DNAfragments may be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to a scFvgene. In these manipulations, a VL- or VH-encoding DNA fragment isoperatively linked to another DNA fragment encoding another protein,such as an antibody constant region or a flexible linker.

The isolated DNA encoding the VH region may be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (CH1, CH2and CH3). The sequences of human heavy chain constant region genes areknown in the art (see, e.g., Kabat, et al. (1991) Sequences of Proteinsof Immunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242) and DNA fragmentsencompassing these regions may be obtained by standard PCRamplification. The heavy chain constant region may be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but most preferablyis an IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene,the VH-encoding DNA may be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region.

The isolated DNA encoding the VL region may be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see, e.g., Kabat, et al.(1991) Sequences of Proteins of Immunological Interest Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242) and DNA fragments encompassing these regions may be obtained bystandard PCR amplification. The light chain constant region may be akappa or lambda constant region, but most preferably is a kappa constantregion.

To create a scFv gene, the VH- and VL-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly-4-Ser)₃, such that the VH and VLsequences may be expressed as a contiguous single-chain protein, withthe VL and VH regions joined by the flexible linker. See, e.g., Bird, etal. (1988) Science 242: 423-426; Huston, et al. (1988) Proc. Natl. Acad.Sci. USA 85: 5879-5883; McCafferty, et al. (1990) Nature 348: 552-554.

Methods of Producing Antibodies and Fragments Thereof.

The present invention also provides methods for producing antibodies andfragments thereof. Methods of producing antibodies are well known tothose of ordinary skill in the art. For example, methods of producingchimeric antibodies are now well known in the art. See, e.g., U.S. Pat.No. 4,816,567; Morrison, et al. (1984) PNAS USA 81: 8651-55; Neuberger,et al. (1985) Nature 314: 268-270; Boulianne, et al. (1984) Nature 312:643-46.

For example, antibodies or antigen binding fragments may be produced bygenetic engineering. In this technique, as with other methods,antibody-producing cells are sensitized to the desired antigen orimmunogen. The messenger RNA isolated from antibody producing cells isused as a template to make cDNA using PCR amplification. A library ofvectors, each containing one heavy chain gene and one light chain generetaining the initial antigen specificity, is produced by insertion ofappropriate sections of the amplified immunoglobulin cDNA into theexpression vectors. A combinatorial library is constructed by combiningthe heavy chain gene library with the light chain gene library. Thisresults in a library of clones which co-express a heavy and light chain(resembling the Fab fragment or antigen binding fragment of an antibodymolecule). The vectors that carry these genes are co-transfected into ahost cell. When antibody gene synthesis is induced in the transfectedhost, the heavy and light chain proteins self-assemble to produce activeantibodies that may be detected by screening with the antigen orimmunogen.

Antibodies, and fragments thereof, of the invention may also be producedby constructing, using conventional techniques well known to those ofordinary skill in the art, an expression vector containing an operon anda DNA sequence encoding an antibody heavy chain in which the DNAsequence encoding the CDRs required for antibody specificity is derivedfrom a non-human cell source, while the DNA sequence encoding theremaining parts of the antibody chain is derived from a human cellsource. Furthermore, the invention relates to vectors, especiallyplasmids, cosmids, viruses, bacteriophages and other vectors common ingenetic engineering, which contain the above-mentioned nucleic acidmolecules of the invention. The nucleic acid molecules contained in thevectors may be linked to regulatory elements that ensure thetranscription in prokaryotic and eukaryotic cells.

Vectors contain elements that facilitate manipulation for the expressionof a foreign protein within the target host cell. Conveniently,manipulation of sequences and production of DNA for transformation isfirst performed in a bacterial host (e.g., E. coli) and usually vectorswill include sequences to facilitate such manipulations, including abacterial origin of replication and appropriate bacterial selectionmarker. Selection markers encode proteins necessary for the survival orgrowth of transformed host cells grown in a selective culture medium.Host cells not transformed with the vector containing the selection genewill not survive in the culture medium. Typical selection genes encodeproteins that confer resistance to antibiotics or other toxins,complement auxotrophic deficiencies, or supply critical nutrients notavailable from complex media. Exemplary vectors and methods fortransformation of yeast are described in the art. See, e.g., Burke, etal. (2000) Methods in Yeast Genetics Cold Spring Harbor LaboratoryPress.

The polypeptide coding sequence of interest may be operably linked totranscriptional and translational regulatory sequences that provide forexpression of the polypeptide in yeast cells. These vector componentsmay include, but are not limited to, one or more of the following: anenhancer element, a promoter, and a transcription termination sequence.Sequences for the secretion of the polypeptide may also be included(e.g., a signal sequence).

Nucleic acids are “operably linked” when placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for asignal sequence is operably linked to DNA for a polypeptide if it isexpressed as a preprotein that participates in the secretion of thepolypeptide; a promoter or enhancer is operably linked to a codingsequence if it affects the transcription of the sequence. Generally,“operably linked” refers broadly to contiguous linked DNA sequences,and, in the case of a secretory leader, contiguous and in reading frame.However, enhancers do not have to be contiguous.

Promoters are untranslated sequences located upstream (5′) to the startcodon of a structural gene (generally within about 100 to 1000 bp) thatcontrol the transcription and translation of particular nucleic acidsequences to which they are operably linked. Such promoters fall intoseveral classes: inducible, constitutive, and repressible promoters(e.g., that increase levels of transcription in response to absence of arepressor). Inducible promoters may initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions (e.g., the presence or absence of a nutrient or achange in temperature.)

A second expression vector may be produced using the same conventionalmeans well known to those of ordinary skill in the art, said expressionvector containing an operon and a DNA sequence encoding an antibodylight chain in which the DNA sequence encoding the CDRs required forantibody specificity is derived from a non-human cell source, preferablya rabbit B-cell source, while the DNA sequence encoding the remainingparts of the antibody chain is derived from a human cell source.

The expression vectors are transfected into a host cell by conventiontechniques well known to those of ordinary skill in the art to produce atransfected host cell, said transfected host cell cultured byconventional techniques well known to those of ordinary skill in the artto produce said antibody polypeptides.

The host cell may be co-transfected with the two expression vectorsdescribed above, the first expression vector containing DNA encoding anoperon and a light chain-derived polypeptide and the second vectorcontaining DNA encoding an operon and a heavy chain-derived polypeptide.The two vectors contain different selectable markers, but preferablyachieve substantially equal expression of the heavy and light chainpolypeptides. Alternatively, a single vector may be used, the vectorincluding DNA encoding both the heavy and light chain polypeptides. Thecoding sequences for the heavy and light chains may comprise cDNA,genomic DNA, or both.

The host cells used to express the antibodies, and fragments thereof,may be either a bacterial cell such as E. coli, or a eukaryotic cell. Amammalian cell of a well-defined type for this purpose, such as amyeloma cell, a Chinese hamster ovary (CHO), a NSO, or a HEK293 cellline may be used.

The general methods by which the vectors may be constructed,transfection methods required to produce the host cell and culturingmethods required to produce the antibodies, and fragments thereof, fromsaid host cells all include conventional techniques. Although preferablythe cell line used to produce the antibody is a mammalian cell line, anyother suitable cell line, such as a bacterial cell line such as an E.coli-derived bacterial strain, or a yeast cell line, may be used.

Similarly, once produced the antibodies may be purified according tostandard procedures in the art, such as for example cross-flowfiltration, ammonium sulphate precipitation, and affinity columnchromatography.

Generation of Antibodies that Bind a VISTA or VISTA Conjugate UsingAnimals

The antibodies of the invention that selectively bind the VISTA andVISTA conjugate may be human monoclonal antibodies. Such humanmonoclonal antibodies directed against a VISTA and VISTA conjugate maybe generated using transgenic or transchromosomic mice carrying parts ofthe human immune system rather than the mouse system. These transgenicand transchromosomic mice include mice referred to herein as the HuMAbMouse® and KM Mouse® respectively, and are collectively referred toherein as “human Ig mice.” The HuMAb Mouse® (Medarex, Inc.) containshuman immunoglobulin gene miniloci that encode unrearranged human heavy(μ and γ) and κ light chain immunoglobulin sequences, together withtargeted mutations that inactivate the endogenous μ and κ c chain loci.See, e.g., Lonberg, et al. (1994) Nature 368(6474): 856-859.Accordingly, the mice exhibit reduced expression of mouse IgM or κ, andin response to immunization, the introduced human heavy and light chaintransgenes undergo class switching and somatic mutation to generate highaffinity human IgGκ monoclonal. Lonberg (1994) Handbook of ExperimentalPharmacology 113: 49-101; Lonberg and Huszar (1995) Intern. Rev.Immunol. 13: 65-93, and Harding and Lonberg (1995) Ann. NY. Acad. Sci.764: 536-546. The preparation and use of the HuMab Mouse®, and thegenomic modifications carried by such mice, is further described inTaylor, et al. (1992) Nucleic Acids Research 20: 6287-6295; Chen, et al.(1993) International Immunology 5: 647-656; Tuaillon, et al. (1993)Proc. Natl. Acad. Sci. USA 90: 3720-3724; Choi, et al. (1993) NatureGenetics 4: 117-123; Chen, et al. (1993) EMBO J. 12: 821-830; Tuaillon,et al. (1994) J. Immunol. 152: 2912-2920; Taylor, et al. (1994)International Immunology 6: 579-591; and Fishwild, et al. (1996) NatureBiotechnology 14: 845-851. See further, U.S. Pat. Nos. 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016;5,814,318; 5,874,299; 5,770,429; and 5,545,807; WO 92/03918, WO93/12227, WO 94/25585; WO 97/13852; WO 98/24884; WO 99/45962; and WO01/14424.

Human anti-VISTA and anti-VISTA-Ig conjugate antibodies (e.g.,antibodies which selectively bind the VISTA and VISTA conjugate) of theinvention may be raised using a mouse that carries human immunoglobulinsequences on transgenes and transchromosomes, such as a mouse thatcarries a human heavy chain transgene and a human light chaintranschromosome. Such mice, referred to herein as “KM Mice®”, aredescribed in detail in WO 02/43478.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and may be used to raiseanti-VISTA and anti-VISTA-Ig conjugate antibodies of the invention. Forexample, an alternative transgenic system referred to as the Xenomouse(Abgenix, Inc.) may be used; such mice are described in, for example,U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and may be used to raiseanti-VISTA and anti-VISTA-Ig conjugate antibodies of the invention. Forexample, mice carrying both a human heavy chain transchromosome and ahuman light chain transchromosome, referred to as “TC mice” may be used.See Tomizuka, et al. (2000) Proc. Natl. Acad. Sci. USA 97: 722-727.Furthermore, cows carrying human heavy and light chain transchromosomeshave been described in the art (Kuroiwa, et al. (2002) NatureBiotechnology 20: 889-894) and may be used to raise anti-VISTA andanti-VISTA-Ig conjugate antibodies of the invention.

Human monoclonal antibodies of the invention may also be prepared usingphage display methods for screening libraries of human immunoglobulingenes. Such phage display methods for isolating human antibodies areestablished in the art. See, for example, U.S. Pat. Nos. 5,223,409;5,403,484; 5,571,698; 5,427,908 5,580,717; 5,969,108; 6,172,197;5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081.

Human monoclonal antibodies of the invention may also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response may be generated upon immunization. See,e.g., U.S. Pat. Nos. 5,476,996 and 5,698,767.

When human Ig mice are used to raise human antibodies of the invention,such mice may be immunized with a purified or enriched preparation ofVISTA and VISTA conjugate polypeptide, as described by Lonberg, et al.(1994) Nature 368(6474): 856-859; Fishwild, et al. (1996) NatureBiotechnology 14: 845-851; WO 98/24884 and WO 01/14424. Preferably, themice will be 6-16 weeks of age upon the first infusion. For example, apurified or recombinant preparation (5-50 μg) of VISTA and VISTAconjugate may be used to immunize the human Ig mice intraperitoneally.

Prior experience with various antigens by others has shown that thetransgenic mice respond when initially immunized intraperitoneally (IP)with antigen in complete Freund's adjuvant, followed by every other weekIP immunizations (up to a total of 6) with antigen in incompleteFreund's adjuvant. However, adjuvants other than Freund's are also foundto be effective. In addition, whole cells in the absence of adjuvant arefound to be highly immunogenic. The immune response may be monitoredover the course of the immunization protocol with plasma samples beingobtained by retroorbital bleeds. The plasma may be screened by ELISA (asdescribed below), and mice with sufficient titers of anti-VISTA oranti-VISTA-Ig human immunoglobulin may be used for fusions. Mice may beboosted intravenously with antigen 3 days before sacrifice and removalof the spleen. It is expected that 2-3 fusions for each immunization mayneed to be performed. Between 6 and 24 mice are typically immunized foreach antigen. Usually both HCo7 and HCo12 strains are used. In addition,both HCo7 and HCo12 transgene may be bred together into a single mousehaving two different human heavy chain transgenes (HCo7/HCo12).Alternatively or additionally, the KM Mouse® strain may be used.

Generation of Hybridomas Producing Human Monoclonal Antibodies of theInvention

To generate hybridomas producing human monoclonal antibodies of theinvention, splenocytes and/or lymph node cells from immunized mice maybe isolated and fused to an appropriate immortalized cell line, such asa mouse myeloma cell line. The resulting hybridomas may be screened forthe production of antigen-specific antibodies. For example, single cellsuspensions of splenic lymphocytes from immunized mice may be fused toone-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells(ATCC, CRL 1580) with 50% PEG. Cells may be plated at approximately2×10⁻⁵ in flat bottom microtiter plate, followed by a two weekincubation in selective medium containing 20% fetal Clone Serum, 18%“653” conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mM sodiumpyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units/mlpenicillin, 50 mg/ml streptomycin, 50 mg/ml gentamycin and 1×HAT (Sigma;the HAT is added 24 hours after the fusion). After approximately twoweeks, cells may be cultured in medium in which the HAT is replaced withHT. Individual wells may then be screened by ELISA for human monoclonalIgM and IgG antibodies. Once extensive hybridoma growth occurs, mediummay be observed usually after 10-14 days. The antibody secretinghybridomas may be replated, screened again, and if still positive forhuman IgG, the monoclonal antibodies may be subcloned at least twice bylimiting dilution. The stable subclones may then be cultured in vitro togenerate small amounts of antibody in tissue culture medium forcharacterization.

To purify human monoclonal antibodies, selected hybridomas may be grownin two-liter spinner-flasks for monoclonal antibody purification.Supernatants may be filtered and concentrated before affinitychromatography with protein A-Sepharose (Pharmacia, Piscataway, N.J.)Eluted IgG may be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution may beexchanged into PBS, and the concentration may be determined by OD280using 1.43 extinction coefficient. The monoclonal antibodies may bealiquoted and stored at −80° C.

Transgenic Animals

The host cells of the invention can also be used to produce non-humantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichVISTA-coding sequences have been introduced. Such host cells can then beused to create non-human transgenic animals in which exogenous VISTAsequences have been introduced into their genome or homologousrecombinant animals in which endogenous VISTA sequences have beenaltered. Such animals are useful for studying the function and/oractivity of a VISTA and for identifying and/or evaluating modulators ofVISTA activity. As used herein, a “transgenic animal” is a non-humananimal, preferably a mammal, more preferably a rodent such as a rat ormouse, in which one or more of the cells of the animal includes atransgene. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, amphibians, and the like.A transgene is exogenous DNA which is integrated into the genome of acell from which a transgenic animal develops and which remains in thegenome of the mature animal, thereby directing the expression of anencoded gene product in one or more cell types or tissues of thetransgenic animal. As used herein, a “homologous recombinant animal” isa non-human animal, preferably a mammal, more preferably a mouse, inwhich an endogenous VISTA gene has been altered by homologousrecombination between the endogenous gene and an exogenous DNA moleculeintroduced into a cell of the animal, e.g., an embryonic cell of theanimal, prior to development of the animal. A transgenic animal of theinvention can be created by introducing a VISTA-encoding nucleic acidinto the male pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. The VISTA cDNA sequence of SEQ IDNO: 1 or 4 can be introduced as a transgene into the genome of anon-human animal. Alternatively, a nonhuman homologue of a human VISTAgene, such as a monkey or rat VISTA gene, can be used as a transgene.Alternatively, a VISTA gene homologue, such as another VISTA familymember, can be isolated based on hybridization to the VISTA cDNAsequences of SEQ ID NO: 1 or 3 and used as a transgene. Intronicsequences and polyadenylation signals can also be included in thetransgene to increase the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to a VISTAtransgene to direct expression of a VISTA polypeptide to particularcells. Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder, et al. U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of a VISTA transgene in its genome and/or expression of VISTAmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene encoding a VISTApolypeptide can further be bred to other transgenic animals carryingother transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a VISTA gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the VISTA gene. The VISTA gene can be a human ormurine gene (e.g., the cDNA of SEQ ID NO: 1 or 3)

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc Natl. Acad. Sci.USA 89:6232-6236. Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. (1991) Science251:1351-1355. If a cre/loxP recombinase system is used to regulateexpression of the transgene, animals containing transgenes encoding boththe Cre recombinase and a selected polypeptide are required. Suchanimals can be provided through the construction of “double” transgenicanimals, e.g., by mating two transgenic animals, one containing atransgene encoding a selected polypeptide and the other containing atransgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, et al. (1997)Nature 385: 810-813; WO 97/07668; and WO 97/07669. In brief, a cell,e.g., a somatic cell, from the transgenic animal can be isolated andinduced to exit the growth cycle and enter G0 phase. The quiescent cellcan then be fused, e.g., through the use of electrical pulses, to anenucleated oocyte from an animal of the same species from which thequiescent cell is isolated. The reconstructed oocyte is then culturedsuch that it develops to the morula or blastocyst stage and thentransferred to pseudopregnant female foster animal. The offspring borneof this female foster animal will be a clone of the animal from whichthe cell, e.g., the somatic cell, is isolated.

Labels

The polypeptides, conjugates, and antibodies described herein may bemodified post-translationally to add effector moieties such as chemicallinkers, detectable moieties such as for example fluorescent dyes,enzymes, substrates, bioluminescent materials, radioactive materials,chemiluminescent moieties, a cytotoxic agent, radioactive materials, orfunctional moieties.

A wide variety of entities, e.g., ligands, may be coupled to theoligonucleotides as known in the art. Ligands may include naturallyoccurring molecules, or recombinant or synthetic molecules. Exemplaryligands include, but are not limited to, avadin, biotin, peptides,peptidomimetics, polylysine (PLL), polyethylene glycol (PEG), mPEG,cationic groups, spermine, spermidine, polyamine, thyrotropin,melanotropin, lectin, glycoprotein, surfactant protein A, mucin,glycosylated polyaminoacids, transferrin, aptamer, immunoglobulins(e.g., antibodies), insulin, transferrin, albumin, sugar, lipophilicmolecules (e.g., steroids, bile acids, cholesterol, cholic acid, andfatty acids), vitamin A, vitamin E, vitamin K, vitamin B, folic acid,B12, riboflavin, biotin, pyridoxal, vitamin cofactors,lipopolysaccharide, hormones and hormone receptors, lectins,carbohydrates, multivalent carbohydrates, radiolabeled markers,fluorescent dyes, and derivatives thereof. See, e.g., U.S. Pat. Nos.6,153,737; 6,172,208; 6,300,319; 6,335,434; 6,335,437; 6,395,437;6,444,806; 6,486,308; 6,525,031; 6,528,631; and 6,559, 279.

Additionally, moieties may be added to the antigen or epitope toincrease half-life in vivo (e.g., by lengthening the time to clearancefrom the blood stream. Such techniques include, for example, adding PEGmoieties (also termed pegilation), and are well-known in the art. SeeU.S. Patent Application Publication No. 2003/0031671.

An antigen, antibody or antigen binding fragment thereof, describedherein may be “attached” to a substrate when it is associated with thesolid label through a non-random chemical or physical interaction. Theattachment may be through a covalent bond. However, attachments need notbe covalent or permanent. Materials may be attached to a label through a“spacer molecule” or “linker group.” Such spacer molecules are moleculesthat have a first portion that attaches to the biological material and asecond portion that attaches to the label. Thus, when attached to thelabel, the spacer molecule separates the label and the biologicalmaterials, but is attached to both. Methods of attaching biologicalmaterial (e.g., label) to a label are well known in the art, and includebut are not limited to chemical coupling.

Detectable Labels

The VISTA and VISTA conjugate described herein may be modifiedpost-translationally to add effector labels such as chemical linkers,detectable labels such as for example fluorescent dyes, enzymes,substrates, bioluminescent materials, radioactive materials, andchemiluminescent labels, or functional labels such as for examplestreptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, andradioactive materials. Further exemplary enzymes include, but are notlimited to, horseradish peroxidase, acetylcholinesterase, alkalinephosphatase, β-galactosidase and luciferase. Further exemplaryfluorescent materials include, but are not limited to, rhodamine,fluorescein, fluorescein isothiocyanate, umbelliferone,dichlorotriazinylamine, phycoerythrin and dansyl chloride. Furtherexemplary chemiluminescent labels include, but are not limited to,luminol. Further exemplary bioluminescent materials include, but are notlimited to, luciferin, luciferase, and aequorin. Further exemplaryradioactive materials include, but are not limited to, bismuth-213(²¹³Bs), carbon-14 (¹⁴C), carbon-11 (¹¹Cu), chlorine-18 (Cl¹⁸),chromium-51 (⁵¹Cr), cobalt-57 (⁵⁷Co), cobalt-60 (⁶⁰Co), copper-64(⁶⁴Cu), copper-67 (⁶⁷Cu), dysprosium-165 (¹⁶⁵Dy), erbium-169 (¹⁶⁹Er),fluorine-18 (¹⁸F), gallium-67 (⁶⁷Ga), gallium-68 (⁶⁸Ga), germanium-68(⁶⁸Ge), holmium-166 (¹⁶⁶Ho), indium-111 (¹¹¹In), iodine-125 (¹²⁵I),iodine-123 (¹²⁴I), iodine-124 (¹²⁴I), iodine-131 (¹³¹I), iridium-192(¹⁹²Ir), iron-59 (⁵⁹Fe), krypton-81 (⁸¹Kr), lead-212 (²¹²Pb),lutetium-177 (¹⁷⁷Lu), molybdenum-99 (⁹⁹Mo), nitrogen-13 (¹³N), oxygen-15(¹⁵O), palladium-103 (¹⁰³Pd), phosphors-32 (³²P), potassium-42, (⁴²K),rhenium 186 (¹⁸⁶Re), rhenium 188 (¹⁸⁸Re), rubidium-81 (⁸¹Rb),rubidium-82 (⁸²Rb), samarium-153 (¹⁵³Sm), selenium-75 (⁷⁵Se), sodium-24(²⁴Na), strontium-82 (⁸²Sr), strontium-89 (⁸⁹Sr), sulfur 35 (³⁵S),technetium-99m (⁹⁹Tc), thallium-201 (²⁰¹Tl), tritium (³H), xenon 133(¹³³Xe), ytterbium 169 (¹⁶⁹Yb), ytterbium-177 (¹⁷⁷Yb), and yttrium-90(⁹⁰Y).

Cytotoxic Agents

For making cytotoxic agents, VISTA polypeptides and VISTA conjugates ofthe invention may be linked, or operatively attached, to toxins usingtechniques that are known in the art. A wide variety of toxins are knownthat may be conjugated to polypeptides or antibodies of the invention.Examples include: numerous useful plant-, fungus- or evenbacteria-derived toxins, which, by way of example, include: various Achain toxins, particularly ricin A chain; ribosome inactivating proteinssuch as saporin or gelonin; alpha-sarcin; aspergillin; restrictocin; andribonucleases such as placental ribonuclease, angiogenic, diphtheriatoxin, or pseudomonas exotoxin. A preferred toxin moiety for use inconnection with the invention is toxin A chain which has been treated tomodify or remove carbohydrate residues, deglycosylated A chain. U.S.Pat. No. 5,776,427.

The VISTA and VISTA conjugates described herein may be conjugated tocytotoxic agents including, but are not limited to, methotrexate,aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine; alkylating agents such as mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU), mitomycin C, lomustine(CCNU), 1-methylnitrosourea, cyclothosphamide, mechlorethamine,busulfan, dibromomannitol, streptozotocin, mitomycin C,cis-dichlorodiamine platinum (II) (DDP) cisplatin and carboplatin(paraplatin); anthracyclines include daunorubicin (formerly daunomycin),doxorubicin (adriamycin), detorubicin, caminomycin, idarubicin,epirubicin, mitoxantrone and bisantrene; antibiotics includedactinomycin (actinomycin D), bleomycin, calicheamicin, mithramycin, andanthramycin (AMC); and antimytotic agents such as the vinca alkaloids,vincristine and vinblastine. Other cytotoxic agents include paclitaxel(TAXOL®), ricin, pseudomonas exotoxin, gemcitabine, cytochalasin B,gramicidin D, ethidium bromide, emetine, etoposide, tenoposide,colchicin, dihydroxy anthracin dione, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol,puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids,mytotane (O,P′-(DDD)), interferons, and mixtures of these cytotoxicagents.

Further cytotoxic agents include, but are not limited to,chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel,gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C,actinomycin D, cyclophosphamide, vincristine, bleomycin, VEGFantagonists, EGFR antagonists, platins, taxols, irinotecan,5-fluorouracil, gemcytabine, leucovorine, steroids, cyclophosphamide,melphalan, vinca alkaloids (e.g., vinblastine, vincristine, vindesineand vinorelbine), mustines, tyrosine kinase inhibitors, radiotherapy,sex hormone antagonists, selective androgen receptor modulators,selective estrogen receptor modulators, PDGF antagonists, TNFantagonists, IL-1 antagonists, interleukins (e.g., IL-12 or IL-2),IL-12R antagonists, Toxin conjugated monoclonal antibodies, tumorantigen specific monoclonal antibodies, Erbitux®, Avastin®, Pertuzumab,anti-CD20 antibodies, Rituxan®, ocrelizumab, ofatumumab, DXL625,Herceptin®, or any combination thereof. Toxic enzymes from plants andbacteria such as ricin, diphtheria toxin and Pseudomonas toxin may beconjugated to the humanized antibodies, or binding fragments thereof, togenerate cell-type-specific-killing reagents. Youle, et al. (1980) Proc.Nat'l Acad. Sci. USA 77: 5483; Gilliland, et al. (1980) Proc. Nat'lAcad. Sci. USA 77: 4539; Krolick, et al. (1980) Proc. Nat'l Acad. Sci.USA 77: 5419. Other cytotoxic agents include cytotoxic ribonucleases.See U.S. Pat. No. 6,653,104.

The VISTA protein described herein may be conjugated to a radionuclidethat emits alpha or beta particles (e.g., radioimmunoconjuagtes). Suchradioactive isotopes include but are not limited to beta-emitters suchas phosphorus-32 (³²P), scandium-47 (⁴⁷Sc), copper-67 (⁶⁷Cu), gallium-67(⁶⁷Ga), yttrium-88 (⁸⁸Y), yttrium-90 (⁹⁰Y), iodine-125 (¹²⁵I),iodine-131 (¹³¹I), samarium-153 (¹⁵³Sm), lutetium-177 (¹⁷⁷Lu),rhenium-186 (¹⁸⁶Re) rhenium-188 (¹⁸⁸Re), and alpha-alpha-emitters suchas astatine-211 (²¹²At) lead-212 (²¹²Pb), bismuth-212 (²¹²Bi),bismuth-213 (²¹³Bi) or actinium-225 (²²⁵Ac).

Methods are known in the art for conjugating a VISTA and VISTA conjugatedescribed herein to a label, such as those methods described by Hunter,et al (1962) Nature 144: 945; David, et al. (1974) Biochemistry 13:1014; Pain, et al. (1981) J. Immunol. Meth. 40: 219; and Nygren (1982)Histochem and Cytochem, 30: 407.

Substrates

The VISTA and VISTA conjugate described herein may be attached to asubstrate. A number of substrates (e.g., solid supports) known in theart are suitable for use with the VISTA and VISTA conjugate describedherein. The substrate may be modified to contain channels or otherconfigurations. See Fung (2004) [Ed.] Protein Arrays: Methods andProtocols Humana Press and Kambhampati (2004) [Ed.] Protein MicroarrayTechnology John Wiley & Sons.

Substrate materials include, but are not limited to acrylics, agarose,borosilicate glass, carbon (e.g., carbon nanofiber sheets or pellets),cellulose acetate, cellulose, ceramics, gels, glass (e.g., inorganic,controlled-pore, modified, soda-lime, or functionalized glass), latex,magnetic beads, membranes, metal, metalloids, nitrocellulose, NYLON®,optical fiber bundles, organic polymers, paper, plastics,polyacryloylmorpholide, poly(4-methylbutene), poly(ethyleneterephthalate), poly(vinyl butyrate), polyacrylamide, polybutylene,polycarbonate, polyethylene, polyethyleneglycol terephthalate,polyformaldehyde, polymethacrylate, polymethylmethacrylate,polypropylene, polysaccharides, polystyrene, polyurethanes,polyvinylacetate, polyvinylchloride, polyvinylidene difluoride (PVDF),polyvinylpyrrolidinone, rayon, resins, rubbers, semiconductor materials,SEPHAROSE®, silica, silicon, styrene copolymers, TEFLON®, and variety ofother polymers.

Substrates need not be flat and can include any type of shape includingspherical shapes (e.g., beads) or cylindrical shapes (e.g., fibers).Materials attached to solid supports may be attached to any portion ofthe solid support (e.g., may be attached to an interior portion of aporous solid support material).

The substrate body may be in the form of a bead, box, column, cylinder,disc, dish (e.g., glass dish, PETRI dish), fiber, film, filter,microtiter plate (e.g., 96-well microtiter plate), multi-bladed stick,net, pellet, plate, ring, rod, roll, sheet, slide, stick, tray, tube, orvial. The substrate may be a singular discrete body (e.g., a singletube, a single bead), any number of a plurality of substrate bodies(e.g, a rack of 10 tubes, several beads), or combinations thereof (e.g.,a tray comprises a plurality of microtiter plates, a column filled withbeads, a microtiter plate filed with beads).

An VISTA and VISTA conjugate may be “attached” to a substrate when it isassociated with the solid substrate through a non-random chemical orphysical interaction. The attachment may be through a covalent bond.However, attachments need not be covalent or permanent. Materials may beattached to a substrate through a “spacer molecule” or “linker group.”Such spacer molecules are molecules that have a first portion thatattaches to the biological material and a second portion that attachesto the substrate. Thus, when attached to the substrate, the spacermolecule separates the substrate and the biological materials, but isattached to both. Methods of attaching biological material (e.g., label)to a substrate are well known in the art, and include but are notlimited to chemical coupling.

Plates, such as microtiter plates, which support and contain thesolid-phase for solid-phase synthetic reactions may be used. Microtiterplates may house beads that are used as the solid-phase. By “particle”or “microparticle” or “nanoparticle” or “bead” or “microbead” or“microsphere” herein is meant microparticulate matter having any of avariety of shapes or sizes. The shape may be generally spherical butneed not be spherical, being, for example, cylindrical or polyhedral. Aswill be appreciated by those in the art, the particles may comprise awide variety of materials depending on their use, including, but notlimited to, cross-linked starch, dextrans, cellulose, proteins, organicpolymers including styrene polymers such as polystyrene andmethylstyrene as well as other styrene co-polymers, plastics, glass,ceramics, acrylic polymers, magnetically responsive materials, colloids,thoriasol, carbon graphite, titanium dioxide, nylon, latex, and TEFLON®.See e.g., “Microsphere Detection Guide” from Bangs Laboratories,Fishers, Ind.

The VISTA and VISTA conjugate described herein may be attached to on anyof the forms of substrates described herein (e.g., bead, box, column,cylinder, disc, dish (e.g., glass dish, PETR1 dish), fiber, film,filter, microtiter plate (e.g., 96-well microtiter plate), multi-bladedstick, net, pellet, plate, ring, rod, roll, sheet, slide, stick, tray,tube, or vial). In particular, particles or beads may be a component ofa gelling material or may be separate components such as latex beadsmade of a variety of synthetic plastics (e.g., polystyrene). The label(e.g., streptavidin) may be bound to a substrate (e.g., bead).

Pharmaceutical Compositions

A “pharmaceutical composition” refers to a chemical or biologicalcomposition suitable for administration to a mammal. Such compositionsmay be specifically formulated for administration via one or more of anumber of routes, including but not limited to buccal, epicutaneous,epidural, inhalation, intraarterial, intracardial,intracerebroventricular, intradermal, intramuscular, intranasal,intraocular, intraperitoneal, intraspinal, intrathecal, intravenous,oral, parenteral, rectally via an enema or suppository, subcutaneous,subdermal, sublingual, transdermal, and transmucosal. In addition,administration may occur by means of injection, powder, liquid, gel,drops, or other means of administration.

As noted such compositions may additionally comprise a desired antigen,e.g., a tumor antigen or another immune modulatory compounds such asToll like receptor agonists, type 1 interferon such as alpha and betainterferons and CD40 agonists such as agonistic CD40 antibodies andantibody fragments, preferably anti-human CD40 agonistic antibodies andantibody fragments or other immune enhancers or suppressors such asPD-L1, PD-L2, CTLA4 fusion proteins and antibodies specific thereto.

In one embodiment, the antigen may be a cancer antigen or a tumorantigen. The terms cancer antigen and tumor antigen are usedinterchangeably and refer to an antigen that is differentially expressedby cancer cells. Therefore, cancer antigens can be exploited todifferentially target an immune response against cancer cells. Cancerantigens may thus potentially stimulate tumor-specific immune responses.Certain cancer antigens are encoded, though not necessarily expressed,by normal cells. Some of these antigens may be characterized as normallysilent (i.e., not expressed) in normal cells, those that are expressedonly at certain stages of differentiation, and those that are temporallyexpressed (e.g., embryonic and fetal antigens). Other cancer antigenscan be encoded by mutant cellular genes such as, for example, oncogenes(e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), orfusion proteins resulting from internal deletions or chromosomaltranslocations. Still other cancer antigens can be encoded by viralgenes such as those carried by RNA and DNA tumor viruses.

Examples of tumor antigens include MAGE, MART-1/Melan-A, gp100,Dipeptidyl peptidase IV (DPPUV), adenosine deaminase-binding protein(ADAbp), cyclophilin b, Colorectal associated antigen(CRC)-C017-1A/GA733, Carcinoembryonic Antigen (CEA) and its antigenicepitopes CAP-1 and CAP-2, etv6, am11, Prostate Specific Antigen (PSA)and its antigenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specificmembrane antigen (PSMA), T-cell receptor/CD3-.zeta. chain, MAGE-familyof tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5,MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12,MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1,MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family of tumor antigens(e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8,GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53,MUC family, HER2/neu, p21ras, RCAS1, α-fetoprotein, ε-cadherin,α-catenin, β-catenin, γ-catenin, p120ctn, gp10.sup.Pmel117, PRAME,NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin,Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viralproducts such as human papilloma virus proteins, Smad family of tumorantigens, Imp-1, PIA, EBV-encoded nuclear antigen (EBNA)-1, brainglycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5,SCP-1 and CT-7, and c-erbB-2.

Cancers or tumors and specific tumor antigens associated with suchtumors (but not exclusively), include acute lymphoblastic leukemia(etv6, am11, cyclophilin b), B cell lymphoma (Ig-idiotype), glioma(E-cadherin, α-catenin, β-catenin, γ-catenin, p120ctn), bladder cancer(p21ras), biliary cancer (p21ras), breast cancer (MUC family, HER2/neu,c-erbB-2), cervical carcinoma (p53, p21ras), colon carcinoma (p21ras,HER2/neu, c-erbB-2, MUC family), colorectal cancer (Colorectalassociated antigen (CRC)-CO17-1A/GA733, APC), choriocarcinoma (CEA),epithelial cell cancer (cyclophilin b), gastric cancer (HER2/neu,c-erbB-2, ga733 glycoprotein), hepatocellular cancer (α-fetoprotein),Hodgkins lymphoma (Imp-1, EBNA-1), lung cancer (CEA, MAGE-3, NY-ESO-1),lymphoid cell-derived leukemia (cyclophilin b), melanoma (p5 protein,gp75, oncofetal antigen, GM2 and GD2 gangliosides, Melan-A/MART-1,cdc27, MAGE-3, p21ras, gp100.sup.Pmel117), myeloma (MUC family, p21ras),non-small cell lung carcinoma (HER2/neu, c-erbB-2), nasopharyngealcancer (Imp-1, EBNA-1), ovarian cancer (MUC family, HER2/neu, c-erbB-2),prostate cancer (Prostate Specific Antigen (PSA) and its antigenicepitopes PSA-1, PSA-2, and PSA-3, PSMA, HER2/neu, c-erbB-2, ga733glycoprotein), renal cancer (HER2/neu, c-erbB-2), squamous cell cancersof the cervix and esophagus (viral products such as human papillomavirus proteins), testicular cancer (NY-ESO-1), and T cell leukemia(HTLV-1 epitopes).

A “pharmaceutical excipient” or a “pharmaceutically acceptableexcipient” is a carrier, usually a liquid, in which an activetherapeutic agent is formulated. In one embodiment of the invention, theactive therapeutic agent is a humanized antibody described herein, orone or more fragments thereof. The excipient generally does not provideany pharmacological activity to the formulation, though it may providechemical and/or biological stability, and release characteristics.Exemplary formulations may be found, for example, in Grennaro (2005)[Ed.] Remington: The Science and Practice of Pharmacy [21^(st) Ed.]

Pharmaceutical compositions typically must be sterile and stable underthe conditions of manufacture and storage. The invention contemplatesthat the pharmaceutical composition is present in lyophilized form. Thecomposition may be formulated as a solution, microemulsion, liposome, orother ordered structure suitable to high drug concentration. The carriermay be a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, and liquidpolyethylene glycol), and suitable mixtures thereof. The inventionfurther contemplates the inclusion of a stabilizer in the pharmaceuticalcomposition.

The polypeptides, conjugates, and antibodies described herein may beformulated into pharmaceutical compositions of various dosage forms. Toprepare the pharmaceutical compositions of the invention, at least oneVISTA and VISTA conjugate as the active ingredient may be intimatelymixed with appropriate carriers and additives according to techniqueswell known to those skilled in the art of pharmaceutical formulations.See Grennaro (2005) [Ed.] Remington: The Science and Practice ofPharmacy [21^(st) Ed.] For example, the antibodies described herein maybe formulated in phosphate buffered saline pH 7.2 and supplied as a 5.0mg/mL clear colorless liquid solution.

Similarly, compositions for liquid preparations include solutions,emulsions, dispersions, suspensions, syrups, and elixirs, with suitablecarriers and additives including but not limited to water, alcohols,oils, glycols, preservatives, flavoring agents, coloring agents, andsuspending agents. Typical preparations for parenteral administrationcomprise the active ingredient with a carrier such as sterile water orparenterally acceptable oil including but not limited to polyethyleneglycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil, withother additives for aiding solubility or preservation may also beincluded. In the case of a solution, it may be lyophilized to a powderand then reconstituted immediately prior to use. For dispersions andsuspensions, appropriate carriers and additives include aqueous gums,celluloses, silicates, or oils.

For each of the recited embodiments, the VISTA and VISTA conjugate maybe administered by a variety of dosage forms. Anybiologically-acceptable dosage form known to persons of ordinary skillin the art, and combinations thereof, are contemplated. Examples of suchdosage forms include, without limitation, reconstitutable powders,elixirs, liquids, solutions, suspensions, emulsions, powders, granules,particles, microparticles, dispersible granules, cachets, inhalants,aerosol inhalants, patches, particle inhalants, implants, depotimplants, injectables (including subcutaneous, intramuscular,intravenous, and intradermal), infusions, and combinations thereof.

In many cases, it will be preferable to include isotonic agents, e.g.,sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions maybe brought about by including in the composition an agent which delaysabsorption, e.g., monostearate salts and gelatin. Moreover, thecompounds described herein may be formulated in a time releaseformulation, e.g. in a composition that includes a slow release polymer.The VISTA and VISTA conjugate may be prepared with carriers that willprotect the compound against rapid release, such as a controlled releaseformulation, including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers may be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers(PLG). Many methods for the preparation of such formulations are knownto those skilled in the art.

Supplementary active compounds can also be incorporated into thecompositions.

For example, compositions may further comprise a desired antigen, e.g.,a tumor antigen or another immune modulatory compounds such as Toll likereceptor agonists, type 1 interferon such as alpha and beta interferonsand CD40 agonists such as agonistic CD40 antibodies and antibodyfragments, preferably anti-human CD40 agonistic antibodies and antibodyfragments or other immune enhancers or suppressors such as PD-L1, PD-L2,CTLA4 fusion proteins and antibodies specific thereto.

Compositions comprising VISTA may further comprise an antigen or otherimmune agonist. The antigen may be administered in an amount that, incombination with the other components of the combination, is effectiveto generate an immune response against the antigen. For example, theantigen may be administered in an amount from about 100 μg/kg to about100 mg/kg. In some embodiments, the antigen may be administered in anamount from about 10 μg/kg to about 10 mg/kg. In some embodiments, theantigen may be administered in an amount from about 1 mg/kg to about 5mg/kg. The particular amount of antigen that constitutes an amounteffective to generate an immune response, however, depends to someextent upon certain factors such as, for example, the particular antigenbeing administered; the particular agonist being administered and theamount thereof; the particular agonist being administered and the amountthereof; the state of the immune system; the method and order ofadministration of the agonist and the antigen; the species to which theformulation is being administered; and the desired therapeutic result.Accordingly, it is not practical to set forth generally the amount thatconstitutes an effective amount of the antigen. Those of ordinary skillin the art, however, can readily determine the appropriate amount withdue consideration of such factors.

The antigen can be any material capable of raising a Th1 immuneresponse, which may include one or more of, for example, a CD8+ T cellresponse, an NK T cell response, a γ/δ T cell response, or a Th1antibody response. Suitable antigens include but are not limited topeptides; polypeptides; lipids; glycolipids; polysaccharides;carbohydrates; polynucleotides; prions; live or inactivated bacteria,viruses or fungi; and bacterial, viral, fungal, protozoal,tumor-derived, or organism-derived antigens, toxins or toxoids.

Furthermore, certain currently experimental antigens, especiallymaterials such as recombinant proteins, glycoproteins, and peptides thatdo not raise a strong immune response, can be used in connection withadjuvant combinations of the invention. Exemplary experimental subunitantigens include those related to viral disease such as adenovirus,AIDS, chicken pox, cytomegalovirus, dengue, feline leukemia, fowlplague, hepatitis A, hepatitis B, HSV-1, HSV-2, hog cholera, influenzaA, influenza B, Japanese encephalitis, measles, parainfluenza, rabies,respiratory syncytial virus, rotavirus, wart, and yellow fever.

The antigen may be a cancer antigen or a tumor antigen. The terms cancerantigen and tumor antigen are used interchangeably and refer to anantigen that is differentially expressed by cancer cells. Therefore,cancer antigens can be exploited to differentially target an immuneresponse against cancer cells. Cancer antigens may thus potentiallystimulate tumor-specific immune responses. Certain cancer antigens areencoded, though not necessarily expressed, by normal cells. Some ofthese antigens may be characterized as normally silent (i.e., notexpressed) in normal cells, those that are expressed only at certainstages of differentiation, and those that are temporally expressed(e.g., embryonic and fetal antigens). Other cancer antigens can beencoded by mutant cellular genes such as, for example, oncogenes (e.g.,activated ras oncogene), suppressor genes (e.g., mutant p53), or fusionproteins resulting from internal deletions or chromosomaltranslocations. Still other cancer antigens can be encoded by viralgenes such as those carried by RNA and DNA tumor viruses.

Examples of tumor antigens include MAGE, MART-1/Melan-A, gp100,Dipeptidyl peptidase IV (DPPUV), adenosine deaminase-binding protein(ADAbp), cyclophilin b, Colorectal associated antigen(CRC)-C017-1A/GA733, Carcinoembryonic Antigen (CEA) and its antigenicepitopes CAP-1 and CAP-2, etv6, am11, Prostate Specific Antigen (PSA)and its antigenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specificmembrane antigen (PSMA), T-cell receptor/CD3-ζ chain, MAGE-family oftumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5,MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12,MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1,MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family of tumor antigens(e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8,GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53,MUC family, HER2/neu, p21ras, RCAS1, α-fetoprotein, ε-cadherin,α-catenin, β-catenin, γ-catenin, p120ctn, gp10.sup.Pmel117, PRAME,NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin,Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viralproducts such as human papilloma virus proteins, Smad family of tumorantigens, Imp-1, PIA, EBV-encoded nuclear antigen (EBNA)-1, brainglycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5,SCP-1 and CT-7, and c-erbB-2.

Cancers or tumors and specific tumor antigens associated with suchtumors (but not exclusively), include acute lymphoblastic leukemia(etv6, am11, cyclophilin b), B cell lymphoma (Ig-idiotype), glioma(E-cadherin, α-catenin, β-catenin, γ-catenin, p120ctn), bladder cancer(p21ras), biliary cancer (p21ras), breast cancer (MUC family, HER2/neu,c-erbB-2), cervical carcinoma (p53, p21ras), colon carcinoma (p21ras,HER2/neu, c-erbB-2, MUC family), colorectal cancer (Colorectalassociated antigen (CRC)-CO17-1A/GA733, APC), choriocarcinoma (CEA),epithelial cell cancer (cyclophilin b), gastric cancer (HER2/neu,c-erbB-2, ga733 glycoprotein), hepatocellular cancer (α-fetoprotein),Hodgkins lymphoma (Imp-1, EBNA-1), lung cancer (CEA, MAGE-3, NY-ESO-1),lymphoid cell-derived leukemia (cyclophilin b), melanoma (p5 protein,gp75, oncofetal antigen, GM2 and GD2 gangliosides, Melan-A/MART-1,cdc27, MAGE-3, p21ras, gp100.sup.Pmel117), myeloma (MUC family, p21ras),non-small cell lung carcinoma (HER2/neu, c-erbB-2), nasopharyngealcancer (Imp-1, EBNA-1), ovarian cancer (MUC family, HER2/neu, c-erbB-2),prostate cancer (Prostate Specific Antigen (PSA) and its antigenicepitopes PSA-1, PSA-2, and PSA-3, PSMA, HER2/neu, c-erbB-2, ga733glycoprotein), renal cancer (HER2/neu, c-erbB-2), squamous cell cancersof the cervix and esophagus (viral products such as human papillomavirus proteins), testicular cancer (NY-ESO-1), and T cell leukemia(HTLV-1 epitopes).

A person of skill in the art would be able to determine an effectivedosage and frequency of administration through routine experimentation,for example guided by the disclosure herein and the teachings inGoodman, et al. (2011) Goodman & Gilman's The Pharmacological Basis ofTherapeutics [12^(th) Ed.]; Howland, et al. (2005) Lippincott'sIllustrated Reviews Pharmacology [2^(nd) Ed.]; and Golan, (2008)Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy[2^(nd) Ed.] See, also, Grennaro (2005) [Ed.] Remington: The Science andPractice of Pharmacy [21^(st) Ed.]

Routes of Administration

The compositions described herein may be administered in any of thefollowing routes: buccal, epicutaneous, epidural, infusion, inhalation,intraarterial, intracardial, intracerebroventricular, intradermal,intramuscular, intranasal, intraocular, intraperitoneal, intraspinal,intrathecal, intravenous, oral, parenteral, pulmonary, rectally via anenema or suppository, subcutaneous, subdermal, sublingual, transdermal,and transmucosal. The preferred routes of administration are intravenousinjection or infusion. The administration can be local, where thecomposition is administered directly, close to, in the locality, near,at, about, or in the vicinity of, the site(s) of disease, e.g., tumor,or systemic, wherein the composition is given to the patient and passesthrough the body widely, thereby reaching the site(s) of disease. Localadministration (e.g., injection) may be accomplished by administrationto the cell, tissue, organ, and/or organ system, which encompassesand/or is affected by the disease, and/or where the disease signs and/orsymptoms are active or are likely to occur (e.g., tumor site).Administration can be topical with a local effect, composition isapplied directly where its action is desired (e.g., tumor site).

For each of the recited embodiments, the compounds can be administeredby a variety of dosage forms as known in the art. Anybiologically-acceptable dosage form known to persons of ordinary skillin the art, and combinations thereof, are contemplated. Examples of suchdosage forms include, without limitation, chewable tablets, quickdissolve tablets, effervescent tablets, reconstitutable powders,elixirs, liquids, solutions, suspensions, emulsions, tablets,multi-layer tablets, bi-layer tablets, capsules, soft gelatin capsules,hard gelatin capsules, caplets, lozenges, chewable lozenges, beads,powders, gum, granules, particles, microparticles, dispersible granules,cachets, douches, suppositories, creams, topicals, inhalants, aerosolinhalants, patches, particle inhalants, implants, depot implants,ingestibles, injectables (including subcutaneous, intramuscular,intravenous, and intradermal), infusions, and combinations thereof.

Other compounds which can be included by admixture are, for example,medically inert ingredients (e.g., solid and liquid diluent), such aslactose, dextrosesaccharose, cellulose, starch or calcium phosphate fortablets or capsules, olive oil or ethyl oleate for soft capsules andwater or vegetable oil for suspensions or emulsions; lubricating agentssuch as silica, talc, stearic acid, magnesium or calcium stearate and/orpolyethylene glycols; gelling agents such as colloidal clays; thickeningagents such as gum tragacanth or sodium alginate, binding agents such asstarches, arabic gums, gelatin, methylcellulose, carboxymethylcelluloseor polyvinylpyrrolidone; disintegrating agents such as starch, alginicacid, alginates or sodium starch glycolate; effervescing mixtures;dyestuff; sweeteners; wetting agents such as lecithin, polysorbates orlaurylsulphates; and other therapeutically acceptable accessoryingredients, such as humectants, preservatives, buffers andantioxidants, which are known additives for such formulations.

Liquid dispersions for oral administration can be syrups, emulsions,solutions, or suspensions. The syrups can contain as a carrier, forexample, saccharose or saccharose with glycerol and/or mannitol and/orsorbitol. The suspensions and the emulsions can contain a carrier, forexample a natural gum, agar, sodium alginate, pectin, methylcellulose,carboxymethylcellulose, or polyvinyl alcohol.

In further embodiments, the present invention provides kits includingone or more containers comprising pharmaceutical dosage units comprisingan effective amount of one or more antibodies and fragments thereof ofthe present invention. Kits may include instructions, directions,labels, marketing information, warnings, or information pamphlets.

Dosages

The amount of VISTA or VISTA conjugate in a therapeutic compositionaccording to any embodiments of this invention may vary according tofactors such as the disease state, age, gender, weight, patient history,risk factors, predisposition to disease, administration route,pre-existing treatment regime (e.g., possible interactions with othermedications), and weight of the individual. Dosage regimens may beadjusted to provide the optimum therapeutic response. For example, asingle bolus may be administered, several divided doses may beadministered over time, or the dose may be proportionally reduced orincreased as indicated by the exigencies of therapeutic situation.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of antibodies, and fragmentsthereof, calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the antibodies, and fragmentsthereof, and the particular therapeutic effect to be achieved, and thelimitations inherent in the art of compounding such an antibodies, andfragments thereof, for the treatment of sensitivity in individuals. Intherapeutic use for treatment of conditions in mammals (e.g., humans)for which the antibodies and fragments thereof of the present inventionor an appropriate pharmaceutical composition thereof are effective, theantibodies and fragments thereof of the present invention may beadministered in an effective amount. The dosages as suitable for thisinvention may be a composition, a pharmaceutical composition or anyother compositions described herein.

The dosage may be administered as a single dose, a double dose, a tripledose, a quadruple dose, and/or a quintuple dose. The dosages may beadministered singularly, simultaneously, and sequentially.

The dosage form may be any form of release known to persons of ordinaryskill in the art. The compositions of the present invention may beformulated to provide immediate release of the active ingredient orsustained or controlled release of the active ingredient. In a sustainedrelease or controlled release preparation, release of the activeingredient may occur at a rate such that blood levels are maintainedwithin a therapeutic range but below toxic levels over an extendedperiod of time (e.g., 4 to 24 hours). The preferred dosage forms includeimmediate release, extended release, pulse release, variable release,controlled release, timed release, sustained release, delayed release,long acting, and combinations thereof, and are known in the art.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The skilled artisan will appreciate that certainfactors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of a protein, polypeptide, orantibody can include a single treatment or, preferably, can include aseries of treatments.

In a preferred example, a subject is treated with antibody, protein, orpolypeptide in the range of between about 0.1 to 20 mg/kg body weight,one time per week for between about 1 to 10 weeks, preferably between 2to 8 weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. It will also be appreciated thatthe effective dosage of antibody, protein, or polypeptide used fortreatment may increase or decrease over the course of a particulartreatment. Changes in dosage may result and become apparent from theresults of diagnostic assays as described herein.

It will be appreciated that the pharmacological activity of thecompositions may be monitored using standard pharmacological models thatare known in the art. Furthermore, it will be appreciated that thecompositions comprising a VISTA and VISTA conjugate, antibody orantigen-binding fragment thereof, may be incorporated or encapsulated ina suitable polymer matrix or membrane for site-specific delivery, or maybe functionalized with specific targeting agents capable of effectingsite specific delivery. These techniques, as well as other drug deliverytechniques are well known in the art. Determination of optimal dosagesfor a particular situation is within the capabilities of those skilledin the art. See, e.g., Grennaro (2005) [Ed.] Remington: The Science andPractice of Pharmacy [21^(St) Ed.]

Methods of Treatment

The VISTA and VISTA conjugates described herein may be used in methodsfor treating inflammatory disorders, autoimmune diseases, suppress CD4⁺T cell proliferation, suppress CD8⁺ T cell proliferation, suppress CD4⁺T cell cytokine production, and suppress CD8⁺ T cell cytokine productioncomprising administering an effective amount of a VISTA and VISTAconjugate to a subject in need thereof. Further, the VISTA and VISTAconjugates described herein may be used to manufacture medicaments foruse in treating autoimmune diseases, suppress CD4⁺ T cell proliferation,suppress CD8⁺ T cell proliferation, suppress CD4⁺ T cell cytokineproduction, and suppress CD8⁺ T cell cytokine production comprising aneffective amount of a VISTA and VISTA conjugate described herein. TheVISTA and VISTA conjugates described herein may be admixed with apharmaceutically acceptable carrier to manufacture a composition fortreating autoimmune diseases, suppress CD4⁺ T cell proliferation,suppress CD8⁺ T cell proliferation, suppress CD4⁺ T cell cytokineproduction, and suppress CD8⁺ T cell cytokine production comprising aneffective amount of a VISTA or VISTA conjugate described herein.

The therapeutic methods described herein may comprise administration ofPD-L3 or VISTA, is a novel and structurally-distinct, Ig-superfamilyinhibitory ligand, whose extracellular domain bears homology to the B7family ligand PD-L1. This molecule is referred to interchangeably hereinas PD-L3 or VISTA or as V-domain Immunoglobulin Suppressor of T cellActivation (VISTA). VISTA is expressed primarily within thehematopoietic compartment and is highly regulated on myeloid APCs and Tcells. Therapeutic intervention of the VISTA inhibitory pathwayrepresents a novel approach to modulate T cell-mediated immunity for thetreatment of a wide variety of cancers. VISTA polypeptides, conjugates,nucleic acids, ligands, and modulators thereof, may be useful inregulating immunity, especially T cell immunity, for the treatment ofautoimmune disorders and inflammatory disorders.

The use of VISTA, VISTA-conjugates (e.g., VISTA-Ig), and anti-VISTAantibodies to treat cancers including but not limited to colorectalcancer, bladder cancer, ovarian cancer, and melanoma, autoimmunedisorders, and inflammatory disorders. In addition, the presentinvention in particular relates to the use of VISTA proteins, especiallymultimeric VISTA proteins and viral vectors (e.g., adenoviral) thatexpress same to treat conditions wherein immunosupression istherapeutically desired such as allergy, autoimmune disorders, andinflammatory conditions.

The patient may express symptoms of an autoimmune disease or a patientwithout symptoms. The methods described herein may be used on cells,e.g., human cells, in vitro or ex vivo. Alternatively, the method may beperformed on cells present in a subject as part of an in vivo (e.g.,therapeutic) protocol.

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disordercharacterized by insufficient or excessive production of VISTA (PD-L3)protein or production of VISTA (PD-L3) protein forms which havedecreased or aberrant activity compared to VISTA (PD-L3) wild typeprotein. Moreover, the anti-VISTA (PD-L3) antibodies of the inventioncan be used to detect and isolate VISTA (PD-L3) proteins, regulate thebioavailability of VISTA (PD-L3) proteins, and modulate VISTA (PD-L3)activity, e.g., by modulating the interaction of VISTA (PD-L3) with itscounter receptor.

Uses and Methods of the Invention

The VISTA molecules, e.g., the VISTA nucleic acid molecules,polypeptides, polypeptide homologues, and antibodies and antibodyfragments described herein can be used in one or more of the followingmethods: a) screening assays; b) predictive medicine (e.g., diagnosticassays, prognostic assays, and monitoring clinical trials); and c)methods of treatment (e.g., therapeutic and prophylactic, e.g., by up-or down-modulating the immune response). As described herein, a VISTA(PD-L3) polypeptide of the invention has one or more of the followingactivities: 1) binds to and/or modulates the activity of its naturalbinding partner(s), 2) modulates intra- or intercellular signaling, 3)modulates activation of T lymphocytes, 4) modulates the immune responseof an organism, e.g., a mammalian organism, such as a mouse or human.The isolated nucleic acid molecules of the invention can be used, forexample, to express VISTA (PD-L3) polypeptide (e.g., via a recombinantexpression vector in a host cell in gene therapy applications), todetect VISTA (PD-L3) mRNA (e.g., in a biological sample) or a geneticalteration in a VISTA (PD-L3) gene, and to modulate VISTA (PD-L3)activity, as described further below. The VISTA (PD-L3) polypeptides canbe used to treat conditions or disorders characterized by insufficientor excessive production of a VISTA (PD-L3) polypeptide or production ofVISTA (PD-L3) inhibitors. In addition, the VISTA (PD-L3) polypeptidescan be used to screen for naturally occurring VISTA (PD-L3) bindingpartner(s), to screen for drugs or compounds which modulate VISTA(PD-L3) activity, as well as to treat conditions or disorderscharacterized by insufficient or excessive production of VISTA (PD-L3)polypeptide or production of VISTA (PD-L3) polypeptide forms which havedecreased, aberrant or unwanted activity compared to VISTA (PD-L3)wild-type polypeptide (e.g., immune system disorders such as severecombined immunodeficiency, multiple sclerosis, systemic lupuserythematosus, type I diabetes mellitus, lymphoproliferative syndrome,inflammatory bowel disease, allergies, asthma, graft-versus-hostdisease, and transplant rejection; immune responses to infectiouspathogens such as bacteria and viruses; and immune system cancers suchas lymphomas and leukemias). Moreover, the anti-VISTA (PD-L3) antibodiesof the invention can be used to detect and isolate VISTA (PD-L3)polypeptides, regulate the bioavailability of VISTA (PD-L3)polypeptides, and modulate VISTA (PD-L3) activity, e.g., by modulatingthe interaction between VISTA (PD-L3) and its natural bindingpartner(s).

Anti-VISTA (PD-L3) antibodies for use as therapeutics may be selectedbased on the fact that in the presence of soluble VISTA (PD-L3)-proteins(e.g., VISTA (PD-L3)-Ig fusion protein), the anti-VISTA antibodiesenhance the suppressive effects of VISTA (PD-L3) on VISTA (PD-L3)related immune functions. This is quite unexpected as these anti-VISTAantibodies behave in vivo opposite to what would be expected from theirin vitro effect on immunity (i.e., these anti-VISTA monoclonalantibodies are immunosuppressive.)

An important aspect of the invention pertains to methods of modulatingVISTA (PD-L3) expression or activity or interaction with its naturalbinding partners, Relevant to therapy VISTA (PD-L3) has beendemonstrated to inhibit CD28 costimulation, to inhibit TCR activation ofimmune cells, to inhibit proliferation of activated immune cells (CD4+and CD8+ T cells), to inhibit cytokine production by T cells (IL-2,gamma interferon) and to transmit an inhibitory signal to immune cells.Accordingly, the activity and/or expression of VISTA (PD-L3), as well asthe interaction between VISTA (PD-L3) and its binding partners) on Tcells can be modulated in order to modulate the immune response. BecauseVISTA (PD-L3) binds to inhibitory receptors (on T cells), upregulationof VISTA (PD-L3) activity should result in downregulation of immuneresponses, whereas downregulation of VISTA (PD-L3) activity shouldresults in upregulation of immune responses. In an embodiment, VISTA(PD-L3) binds to inhibitory receptors. As noted previously,counterintuitively VISTA (PD-L3) specific antibodies produced byApplicant which in vitro (in the presence of VISTA (PD-L3)-Ig) enhancethe suppressive activities of VISTA (PD-L3)-Ig fusion proteins (i.e.,these antibodies enhance the suppression of VISTA (PD-L3) relatedactivities such as effects of VISTA (PD-L3) on cytokine production, Tcell proliferation, differentiation or activation and other functionsnoted previously), behave oppositely to what would be expected in vivo,i.e., these antibodies have been found to be immunosuppressive in vivo.

Modulatory methods of the invention involve contacting a cell with aVISTA (PD-L3) polypeptide or agent that modulates one or more of theactivities of VISTA (PD-L3) polypeptide activity associated with thecell, e.g., an agent that modulates expression or activity of VISTA(PD-L3) and/or modulates the interaction of VISTA (PD-L3) and itsnatural binding partner(s). An agent that modulates VISTA (PD-L3)polypeptide activity can be an agent as described herein, such as anucleic acid or a polypeptide, a naturally-occurring binding partner ofa VISTA (PD-L3) polypeptide a VISTA (PD-L3) antibody, a VISTA (PD-L3)agonist or antagonist, a peptidomimetic of a VISTA (PD-L3) agonist orantagonist, a VISTA (PD-L3) peptidomimetic, or other small molecule.Soluble forms of VISTA (PD-L3) may also be used to interfere with thebinding of VISTA (PD-L3) to any of its natural binding partner(s) orligands.

An agent that modulates the expression of VISTA (PD-L3) is, e.g., anantisense nucleic acid molecule, triplex oligonucleotide, ribozyme, orrecombinant vector for expression of a VISTA (PD-L3) polypeptide. Forexample, an oligonucleotide complementary to the area around a VISTA(PD-L3) polypeptide translation initiation site can be synthesized. Oneor more antisense oligonucleotides can be added to cell media, typicallyat 200 μg/ml, or administered to a patient to prevent the synthesis of aVISTA (PD-L3) polypeptide. The antisense oligonucleotide is taken up bycells and hybridizes to a VISTA (PD-L3) mRNA to prevent translation.Alternatively, an oligonucleotide which binds double-stranded DNA toform a triplex construct to prevent DNA unwinding and transcription canbe used. As a result of either, synthesis of VISTA (PD-L3) polypeptideis blocked. When VISTA (PD-L3) expression is modulated, preferably, suchmodulation occurs by a means other than by knocking out the VISTA(PD-L3) gene.

Agents which modulate expression, by virtue of the fact that theycontrol the amount of VISTA (PD-L3) in a cell, also modulate the totalamount of VISTA (PD-L3) activity in a cell. In one embodiment, the agentthe modulates VISTA (PD-L3) stimulates one or more VISTA (PD-L3)activities. Examples of such stimulatory agents include active VISTA(PD-L3) polypeptide and a nucleic acid molecule encoding VISTA (PD-L3)that has been introduced into the cell. In another embodiment, the agentinhibits one or more VISTA (PD-L3) activities. Examples of suchinhibitory agents include antisense VISTA (PD-L3) nucleic acidmolecules, anti-VISTA (PD-L3) antibodies, VISTA (PD-L3) inhibitors, andcompounds identified in the subject screening assays. In a furtherembodiment, an inhibitory agent is a combination of an anti-VISTA(PD-L3) antibody and an anti-PD-L1 or anti-PD-L2 antibody. Thesemodulatory methods can be performed in vitro (e.g., by contacting thecell with the agent) or, alternatively, by contacting an agent withcells in vivo (e.g., by administering the agent to a subject). As such,the present invention provides methods of treating an individualafflicted with a condition or disorder that would benefit from up- ordown-modulation of a VISTA (PD-L3) polypeptide, e.g., a disordercharacterized by unwanted, insufficient, or aberrant expression oractivity of a VISTA (PD-L3) polypeptide or nucleic acid molecule. In oneembodiment, the method involves administering an agent (e.g., an agentidentified by a screening assay described herein), or combination ofagents that modulates (e.g., upregulates or downregulates) VISTA (PD-L3)expression or activity. In another embodiment, the method involvesadministering a VISTA (PD-L3) polypeptide or nucleic acid molecule astherapy to compensate for reduced, aberrant, or unwanted VISTA (PD-L3)expression or activity.

The invention provides a method for preventing in a subject, a diseaseor condition associated with an aberrant or unwanted VISTA (PD-L3)expression or activity, by administering to the subject a VISTA (PD-L3)polypeptide or an agent which modulates VISTA (PD-L3) expression or atleast one VISTA (PD-L3) activity. Subjects at risk for a disease ordisorder which is caused or contributed to by aberrant or unwanted VISTA(PD-L3) expression or activity can be identified by, for example, any ora combination of diagnostic or prognostic assays as described herein.Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of the VISTA (PD-L3) aberrancy,such that a disease or disorder is prevented or, alternatively, delayedin its progression. Depending on the type of VISTA (PD-L3) aberrancy,for example, a VISTA (PD-L3) polypeptide, VISTA (PD-L3) agonist or VISTA(PD-L3) antagonist (e.g., an anti-VISTA (PD-L3) antibody) agent can beused for treating the subject. The appropriate agent can be determinedbased on screening assays described herein.

The VISTA and VISTA conjugate, may be admixed with additionalchemotherapeutic agents, cytotoxic agent, antibodies (e.g., anti-PD-L1,PD-L2 or CTLA-4 antibodies), lymphokine, or hematopoietic growth factor.The VISTA and VISTA conjugate, may also be administered in combinationwith another antibody, a lymphokine, cytotoxic agent (e.g., a moietythat inhibits DNA, RNA, or protein synthesis, a radionuclide, orribosomal inhibiting protein, e.g., ²¹²Bi, ¹³¹I, ¹⁸⁸Re, ⁹⁰Y, vindesine,methotrexate, adriamycin, cisplatin, pokeweed antiviral protein,Pseudomonas exotoxin A, ricin, diphtheria toxin, ricin A chain, orcytotoxic phospholipase enzyme), immunosuppressive agent (e.g.,cyclosporine, leflunomide, methotrexate, azothiprine, mercaptopurine,dactinomycin, tacrolimus, or sirolimus) or a hematopoietic growthfactor. The VISTA and VISTA conjugate, may be label with achemiluminescent label, paramagnetic label (e.g., aluminum, manganese,platinum, oxygen, lanthanum, lutetium, scandium, yttrium, or gallium),an MRI contrast agent, fluorescent label, bioluminescent label, orradioactive label. In the methods described herein, the second agent maybe administered simultaneously or sequentially with the antibody. Forexample, the second agent may be an agent that downregulates an immuneresponse (e.g., PD-L1, PD-L2 or CTLA-4 fusion protein or antibodyspecific thereto.)

In one embodiment, methods of treating a subject with an autoimmunedisease comprising administering a VISTA and VISTA conjugate, to asubject who may be receiving secondary therapy. Examples of secondarytherapy include chemotherapy, radiotherapy, immunotherapy, phototherapy,cryotherapy, toxin therapy, hormonal therapy, or surgery. Thus, theinvention contemplates use of the methods and compositions inconjunction with standard anti-cancer therapies. The patient to betreated may be of any age. One of skill in the art will recognize thepresence and development of other anticancer therapies which may be usedin conjugation with the VISTA or VISTA conjugate.

Determination of dose is within the level of ordinary skill in the art.The VISTA and VISTA conjugate, may be administered for acute treatment,over one week or less, often over a period of one to three days or maybe used in chronic treatment, over several months or years. In general,a therapeutically effective amount of the VISTA and VISTA conjugate isan amount sufficient to produce a clinically significant change in theautoimmune disease.

An inhibitory signal as transduced by an inhibitory receptor can occureven if a costimulatory receptor (e.g., CD28 or ICOS) in not present onthe immune cell and, thus, is not simply a function of competitionbetween inhibitory receptors and costimulatory receptors for binding ofcostimulatory molecules (Fallarino, et al. (1998) J. Exp. Med. 188:205). Transmission of an inhibitory signal to an immune cell can resultin unresponsiveness, anergy or programmed cell death in the immune cell.Preferably, transmission of an inhibitory signal operates through amechanism that does not involve apoptosis.

Autoimmune Diseases

The VISTA polypeptides, multimeric VISTA polypeptides, VISTA fusionproteins (e.g., VISTA-Ig), and anti-VISTA antibodies described hereinmay be used in compositions, uses, and methods for the treatment ofautoimmune diseases.

V-domain Immunoglobulin containing Suppressor of T cell Activation(VISTA) is a member of a family related to the Immunoglobulin (Ig)superfamily, which exerts profound impact on the immune system. The Igsuperfamily consists of many critical immune regulators, such as the B7family ligands and receptors. The best characterized costimulatoryligands are B7.1 and B7.2 that belong to the Ig superfamily and areexpressed on professional APCs and whose receptors are CD28 and CTLA-4.

The B7 family ligands have expanded to include co-stimulatory B7-H2(ICOS Ligand) and B7-H3, as well as co-inhibitory B7-H1 (PD-L1), B7-DC(PD-L2), B7-H4 (B7S1 or B7x), and B7-H6. Brandt, et al. (2009) J Exp Med206, 1495-1503; Greenwald, et al. (2005) Annu Rev Immunol 23: 515-548.Accordingly, additional CD28 family receptors have been identified. ICOSis expressed on activated T cells and binds to B7-H2. ICOS is a positiveco-regulator, important for T-cell activation, differentiation andfunction. Dong, et al. (2001) Nature 409, 97-101. On the other hand,programmed death 1 (PD-1) negatively regulates T cell responses.PD-1^(−/−) mice develop lupus-like autoimmune disease, or autoimmunedilated cardiomyopathy. Nishimura, et al. (2001) Science 291: 319-322.Recently, CD80 was identified as a second receptor for PD-L1 thattransduces inhibitory signals into T cells. Butte, et al. (2007)Immunity 27, 111-122. The two inhibitory B7 family ligands, PD-L1 andPD-L2, have distinct expression patterns. PD-L2 is expressed induciblyon DCs and macrophages, whereas PD-L1 is broadly expressed on bothhematopoietic cells and non-hematopoietic cell types. Consistent withthe immune-suppressive role of PD-1 receptor, studies using PD-1^(−/−)and PD-L2^(−/−) mice have shown that both ligands have overlapping rolesin inhibiting T-cell proliferation and cytokine production. At thistime, VISTA appears to be selectively expressed hematopoietic cells,which distinguishes it from PD-L1 in distribution, and likely plays acritical role in negatively regulating the development of autoimmunedisease.

A novel and structurally-distinct, Ig-superfamily inhibitory ligand,whose extracellular domain bears highest homology to the B7 familyligand PD-L1. Although its closest relative phylogenetically is PD-L1,it was not designated a PD-L name due to its modest level of similarity(20%). It has a 93 aa cytoplasmic domain with no obvious signaltransducing motifs, except a possible protein kinase C binding site. SeeFIG. 4. VISTA is a negative, regulatory ligand and that is based on thefollowing facts:

A soluble VISTA-Ig fusion protein suppresses in vitro CD4 and CD8 T cellproliferation and cytokine production. Suppression is observed withPD-1^(−/−) T cells indicating that PD-1 is not the VISTA receptor.

Overexpression of VISTA on APCs suppresses in vitro CD4⁺ and CD8⁺ T cellproliferation.

VISTA over-expression on tumor cells impaired protective anti-tumorimmunity in tumor-vaccinated hosts.

VISTA^(−/−) mice develop an inflammatory phenotype, establishing thatVISTA has an immunosuppressive function. VISTA^(−/−) DC stimulate more Tcell proliferation then WT DCs.

Anti-VISTA monoclonal antibody (13F3) blocked VISTA-induced suppressionof T cell responses by VISTA⁺ APCs in vitro to enhance T cellactivation.

Anti-VISTA monoclonal antibody exacerbated EAE and increased thefrequency of encephalitogenic Th17s in vivo.

Anti-VISTA monoclonal antibody induces tumor remission in multiple (6)murine tumor models and VISTA expression on myeloid derived suppressorcells (MDSC) in these models is extremely high, suggesting that VISTA⁺MDSC suppress tumor specific immunity.

The VISTA polypeptides, multimeric VISTA polypeptides, VISTA fusionproteins (e.g., VISTA-Ig), siRNA molecules consisting of any one of thenucleic acid sequences SEQ ID NO: 38-67, and anti-VISTA antibodiesdescribed herein may be used in compositions, uses, and methods for thetreatment of autoimmune diseases or disorders. Examples of autoimmunediseases or disorders include, but are not limited to acquired immunedeficiency syndrome (AIDS), acquired spenic atrophy, acute anterioruveitis, Acute Disseminated Encephalomyelitis (ADEM), acute goutyarthritis, acute necrotizing hemorrhagic leukoencephalitis, acute orchronic sinusitis, acute purulent meningitis (or other central nervoussystem inflammatory disorders), acute serious inflammation, Addison'sdisease, adrenalitis, adult onset diabetes mellitus (Type II diabetes),adult-onset idiopathic hypoparathyroidism (AOIH), Agammaglobulinemia,agranulocytosis, vasculitides, including vasculitis (including largevessel vasculitis (including polymyalgia rheumatica and giant cell(Takayasu's) arthritis), allergic conditions, allergic contactdermatitis, allergic dermatitis, allergic granulomatous angiitis,allergic hypersensitivity disorders, allergic neuritis, allergicreaction, alopecia greata, alopecia totalis, Alport's syndrome,alveolitis (e.g., allergic alveolitis and fibrosing alveolitis),Alzheimer's disease, amyloidosis, amylotrophic lateral sclerosis (ALS;Lou Gehrig's disease), an eosinophil-related disorder (e.g.,eosinophilia), anaphylaxis, ankylosing spondylitis, antgiectasis,antibody-mediated nephritis, Anti-GBM/Anti-TBM nephritis,antigen-antibody complex-mediated diseases, antiglomerular basementmembrane disease, anti-phospholipid antibody syndrome, antiphospholipidsyndrome (APS), aphthae, aphthous stomatitis, aplastic anemia,arrhythmia, arteriosclerosis, arteriosclerotic disorders, arthritis(e.g., rheumatoid arthritis such as acute arthritis, chronic rheumatoidarthritis), arthritis chronica progrediente, arthritis deformans,ascariasis, aspergilloma (or granulomas containing eosinophils),aspergillosis, aspermiogenese, asthma (e.g., asthma bronchiale,bronchial asthma, and auto-immune asthma), ataxia telangiectasia, ataxicsclerosis, atherosclerosis, autism, autoimmune angioedema, autoimmuneaplastic anemia, autoimmune atrophic gastritis, autoimmune diabetes,autoimmune disease of the testis and ovary including autoimmune orchitisand oophoritis, autoimmune disorders associated with collagen disease,autoimmune dysautonomia, autoimmune ear disease (e.g., autoimmune innerear disease (AGED)), autoimmune endocrine diseases including thyroiditissuch as autoimmune thyroiditis, autoimmune enteropathy syndrome,autoimmune gonadal failure, autoimmune hearing loss, autoimmunehemolysis, Autoimmune hepatitis, autoimmune hepatological disorder,autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune innerear disease (AIED), autoimmune myocarditis, autoimmune neutropenia,autoimmune pancreatitis, autoimmune polyendocrinopathies, autoimmunepolyglandular syndrome type I, autoimmune retinopathy, autoimmunethrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmuneurticaria, autoimmune-mediated gastrointestinal diseases, Axonal &neuronal neuropathies, Balo disease, Behcet's disease, benign familialand ischemia-reperfusion injury, benign lymphocytic angiitis, Berger'sdisease (IgA nephropathy), bird-fancier's lung, blindness, Boeck'sdisease, bronchiolitis obliterans (non-transplant) vs NSIP, bronchitis,bronchopneumonic aspergillosis, Bruton's syndrome, bullous pemphigoid,Caplan's syndrome, Cardiomyopathy, cardiovascular ischemia, Castleman'ssyndrome, Celiac disease, celiac sprue (gluten enteropathy), cerebellardegeneration, cerebral ischemia, and disease accompanyingvascularization, Chagas disease, channelopathies (e.g., epilepsy),channelopathies of the CNS, chorioretinitis, choroiditis, an autoimmunehematological disorder, chronic active hepatitis or autoimmune chronicactive hepatitis, chronic contact dermatitis, chronic eosinophilicpneumonia, chronic fatigue syndrome, chronic hepatitis, chronichypersensitivity pneumonitis, chronic inflammatory arthritis, Chronicinflammatory demyelinating polyneuropathy (CIDP), chronic intractableinflammation, chronic mucocutaneous candidiasis, chronic neuropathy(e.g., IgM polyneuropathies or IgM-mediated neuropathy), chronicobstructive airway disease, chronic pulmonary inflammatory disease,Chronic recurrent multifocal ostomyelitis (CRMO), chronic thyroiditis(Hashimoto's thyroiditis) or subacute thyroiditis, Churg-Strausssyndrome, cicatricial pemphigoid/benign mucosal pemphigoid, CNSinflammatory disorders, CNS vasculitis, Coeliac disease, Coganssyndrome, cold agglutinin disease, colitis polyposa, colitis such asulcerative colitis, colitis ulcerosa, collagenous colitis, conditionsinvolving infiltration of T cells and chronic inflammatory responses,congenital heart block, congenital rubella infection, Coombs positiveanemia, coronary artery disease, Coxsackie myocarditis, CREST syndrome(calcinosis, Raynaud's phenomenon), Crohn's disease, cryoglobulinemia,Cushing's syndrome, cyclitis (e.g., chronic cyclitis, heterochroniccyclitis, iridocyclitis, or Fuch's cyclitis), cystic fibrosis,cytokine-induced toxicity, deafness, degenerative arthritis,demyelinating diseases (e.g., autoimmune demyelinating diseases),demyelinating neuropathies, dengue, dermatitis herpetiformis and atopicdermatitis, dermatitis including contact dermatitis, dermatomyositis,dermatoses with acute inflammatory components, Devic's disease(neuromyelitis optica), diabetic large-artery disorder, diabeticnephropathy, diabetic retinopathy, Diamond Blackfan anemia, diffuseinterstitial pulmonary fibrosis, dilated cardiomyopathy, discoid lupus,diseases involving leukocyte diapedesis, Dressler's syndrome,Dupuytren's contracture, echovirus infection, eczema including allergicor atopic eczema, encephalitis such as Rasmussen's encephalitis andlimbic and/or brainstem encephalitis, encephalomyelitis (e.g., allergicencephalomyelitis or encephalomyelitis allergica and experimentalallergic encephalomyelitis (EAE)), endarterial hyperplasia,endocarditis, endocrine ophthamopathy, endometriosis. endomyocardialfibrosis, endophthalmia phacoanaphylactica, endophthalmitis, enteritisallergica, eosinophilia-myalgia syndrome, eosinophilic faciitis,epidemic keratoconjunctivitis, epidermolisis bullosa acquisita (EBA),episclera, episcleritis, Epstein-Barr virus infection, erythema elevatumet diutinum, erythema multiforme, erythema nodosum leprosum, erythemanodosum, erythroblastosis fetalis, esophageal dysmotility, Essentialmixed cryoglobulinemia, ethmoid, Evan's syndrome, Experimental AllergicEncephalomyelitis (EAE), Factor VIII deficiency, farmer's lung, febrisrheumatica, Felty's syndrome, fibromyalgia, fibrosing alveolitis,flariasis, focal segmental glomerulosclerosis (FSGS), food poisoning,frontal, gastric atrophy, giant cell arthritis (temporal arthritis),giant cell hepatitis, giant cell polymyalgia, glomerulonephritides,glomerulonephritis (GN) with and without nephrotic syndrome such aschronic or acute glomerulonephritis (e.g., primary GN), Goodpasture'ssyndrome, gouty arthritis, granulocyte transfusion-associated syndromes,granulomatosis including lymphomatoid granulomatosis, granulomatosiswith polyangiitis (GPA), granulomatous uveitis, Grave's disease,Guillain-Barre syndrome, gutatte psoriasis, haemoglobinuriaparoxysmatica, Hamman-Rich's disease, Hashimoto's disease, Hashimoto'sencephalitis, Hashimoto's thyroiditis, hemochromatosis, hemolytic anemiaor immune hemolytic anemia including autoimmune hemolytic anemia (AIHA),hemolytic anemia, hemophilia A, Henoch-Schonlein purpura, Herpesgestationis, human immunodeficiency virus (HIV) infection, hyperalgesia,hypogammaglobulinemia, hypogonadism, hypoparathyroidism, idiopathicdiabetes insipidus, idiopathic facial paralysis, idiopathichypothyroidism, idiopathic IgA nephropathy, idiopathic membranous GN oridiopathic membranous nephropathy, idiopathic nephritic syndrome,idiopathic pulmonary fibrosis, idiopathic sprue, Idiopathicthrombocytopenic purpura (ITP), IgA nephropathy, IgE-mediated diseases(e.g., anaphylaxis and allergic and atopic rhinitis), IgG4-relatedsclerosing disease, ileitis regionalis, immune complex nephritis, immuneresponses associated with acute and delayed hypersensitivity mediated bycytokines and T-lymphocytes, immune-mediated GN, immunoregulatorylipoproteins, including adult or acute respiratory distress syndrome(ARDS), Inclusion body myositis, infectious arthritis, infertility dueto antispermatozoan antobodies, inflammation of all or part of the uvea,inflammatory bowel disease (IBD) inflammatory hyperproliferative skindiseases, inflammatory myopathy, insulin-dependent diabetes (type 1),insulitis, Interstitial cystitis, interstitial lung disease,interstitial lung fibrosis, iritis, ischemic re-perfusion disorder,joint inflammation, Juvenile arthritis, juvenile dermatomyositis,juvenile diabetes, juvenile onset (Type I) diabetes mellitus, includingpediatric insulin-dependent diabetes mellitus (IDDM), juvenile-onsetrheumatoid arthritis, Kawasaki syndrome, keratoconjunctivitis sicca,kypanosomiasis, Lambert-Eaton syndrome, leishmaniasis, leprosy,leucopenia, leukocyte adhesion deficiency, Leukocytoclastic vasculitis,leukopenia, lichen planus, lichen sclerosus, ligneous conjunctivitis,linear IgA dermatosis, Linear IgA disease (LAD), Loffler's syndrome,lupoid hepatitis, lupus (including nephritis, cerebritis, pediatric,non-renal, extra-renal, discoid, alopecia), Lupus (SLE), lupuserythematosus disseminatus, Lyme arthritis, Lyme disease, lymphoidinterstitial pneumonitis, malaria, male and female autoimmuneinfertility, maxillary, medium vessel vasculitis (including Kawasaki'sdisease and polyarteritis nodosa), membrano- or membranous proliferativeGN (MPGN), including Type I and Type II, and rapidly progressive GN,membranous GN (membranous nephropathy), Meniere's disease, meningitis,microscopic colitis, microscopic polyangiitis, migraine, minimal changenephropathy, Mixed connective tissue disease (MCTD), mononucleosisinfectiosa, Mooren's ulcer, Mucha-Habermann disease, multifocal motorneuropathy, multiple endocrine failure, multiple organ injury syndromesuch as those secondary to septicemia, trauma or hemorrhage, multipleorgan injury syndrome, multiple sclerosis (MS) such as spino-optical MS,multiple sclerosis, mumps, muscular disorders, myasthenia gravis such asthymoma-associated myasthenia gravis, myasthenia gravis, myocarditis,myositis, narcolepsy, necrotizing enterocolitis, and transmural colitis,and autoimmune inflammatory bowel disease, necrotizing, cutaneous, orhypersensitivity vasculitis, neonatal lupus syndrome (NLE), nephrosis,nephrotic syndrome, neurological disease, neuromyelitis optica(Devic's), neuromyelitis optica, neuromyotonia, neutropenia,non-cancerous lymphocytosis, nongranulomatous uveitis, non-malignantthymoma, ocular and orbital inflammatory disorders, ocular cicatricialpemphigoid, oophoritis, ophthalmia symphatica, opsoclonus myoclonussyndrome (OMS), opsoclonus or opsoclonus myoclonus syndrome (OMS), andsensory neuropathy, optic neuritis, orchitis granulomatosa,osteoarthritis, palindromic rheumatism, pancreatitis, pancytopenia,PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated withStreptococcus), paraneoplastic cerebellar degeneration, paraneoplasticsyndrome, paraneoplastic syndromes, including neurologic paraneoplasticsyndromes (e.g., Lambert-Eaton myasthenic syndrome or Eaton-Lambertsyndrome), parasitic diseases such as Lesihmania, paroxysmal nocturnalhemoglobinuria (PNH), Parry Romberg syndrome, pars planitis (peripheraluveitis), Parsonnage-Turner syndrome, parvovirus infection, pemphigoidsuch as pemphigoid bullous and skin pemphigoid, pemphigus (includingpemphigus vulgaris), pemphigus erythematosus, pemphigus foliaceus,pemphigus mucus-membrane pemphigoid, pemphigus, peptic ulcer, periodicparalysis, peripheral neuropathy, perivenous encephalomyelitis,pernicious anemia (anemia perniciosa), pernicious anemia, phacoantigenicuveitis, pneumonocirrhosis, POEMS syndrome, polyarteritis nodosa, TypeI, II, & III, polyarthritis chronica primaria, polychondritis (e.g.,refractory or relapsed polychondritis), polyendocrine autoimmunedisease, polyendocrine failure, polyglandular syndromes (e.g.,autoimmune polyglandular syndromes (or polyglandular endocrinopathysyndromes)), polymyalgia rheumatica, polymyositis,polymyositis/dermatomyositis, polyneuropathies, polyradiculitis acuta,post-cardiotomy syndrome, posterior uveitis, or autoimmune uveitis,postmyocardial infarction syndrome, postpericardiotomy syndrome,post-streptococcal nephritis, post-vaccination syndromes, preseniledementia, primary biliary cirrhosis, primary hypothyroidism, primaryidiopathic myxedema, primary lymphocytosis, which includes monoclonal Bcell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonalgarnmopathy of undetermined significance, MGUS), primary myxedema,primary progressive MS (PPMS), and relapsing remitting MS (RRMS),primary sclerosing cholangitis, progesterone dermatitis, progressivesystemic sclerosis, proliferative arthritis, psoriasis such as plaquepsoriasis, psoriasis, psoriatic arthritis, pulmonary alveolarproteinosis, pulmonary infiltration eosinophilia, pure red cell anemiaor aplasia (PRCA), pure red cell aplasia, purulent or nonpurulentsinusitis, pustular psoriasis and psoriasis of the nails, pyelitis,pyoderma gangrenosum, Quervain's thyreoiditis, Raynauds phenomenon,reactive arthritis, recurrent abortion, reduction in blood pressureresponse, reflex sympathetic dystrophy, refractory sprue, Reiter'sdisease or syndrome, relapsing polychondritis, reperfusion injury ofmyocardial or other tissues, reperfusion injury, respiratory distresssyndrome, restless legs syndrome, retinal autoimmunity, retroperitonealfibrosis, Reynaud's syndrome, rheumatic diseases, rheumatic fever,rheumatism, rheumatoid arthritis, rheumatoid spondylitis, rubella virusinfection, Sampter's syndrome, sarcoidosis, schistosomiasis, Schmidtsyndrome, SCID and Epstein-Barr virus-associated diseases, sclera,scleritis, sclerodactyl, scleroderma (including systemic scleroderma),sclerosing cholangitis, sclerosis disseminata, sclerosis such assystemic sclerosis, sensoneural hearing loss, seronegativespondyloarthritides, Sheehan's syndrome, Shulman's syndrome, silicosis,Sjogren's syndrome, sperm & testicular autoimmunity, sphenoid sinusitis,Stevens-Johnson syndrome, stiff-man (or stiff-person) syndrome, subacutebacterial endocarditis (SBE), subacute cutaneous lupus erythematosus,sudden hearing loss, Susac's syndrome, Sydenham's chorea, sympatheticophthalmia, systemic lupus erythematosus (SLE) or systemic lupuserythematodes (e.g., cutaneous SLE), systemic necrotizing vasculitis,and ANCA-associated vasculitis, such as Churg-Strauss vasculitis orsyndrome (CSS)), tabes dorsalis, Takayasu's arteritis, telangiectasia,temporal arteritis/Giant cell arteritis, thromboangitis ubiterans,thrombocytopenia (as developed by myocardial infarction patients, forexample), including thrombotic thrombocytopenic purpura (TTP) andautoimmune or immune-mediated thrombocytopenia such as idiopathicthrombocytopenic purpura (ITP) including chronic or acute ITP,thrombocytopenic purpura (TTP), thyrotoxicosis, tissue injury,Tolosa-Hunt syndrome, toxic epidermal necrolysis, toxic-shock syndrome,transfusion reaction, transient hypogammaglobulinemia of infancy,transverse myelitis, traverse myelitis, tropical pulmonary eosinophilia,tuberculosis, ulcerative colitis, undifferentiated connective tissuedisease (UCTD), urticaria (e.g., chronic allergic urticaria and chronicidiopathic urticaria, including chronic autoimmune urticaria), uveitis(e.g., anterior uveitis), uveoretinitis, valvulitis, vasculardysfunction, vasculitis, vertebral arthritis, vesiculobullousdermatosis, vitiligo, Wegener's granulomatosis (now termedGranulomatosis with Polyangiitis (GPA), Wiskott-Aldrich syndrome, andx-linked hyper IgM syndrome.

Treatment of Cancer

The VISTA polypeptides, multimeric VISTA polypeptides, VISTA fusionproteins (e.g., VISTA-Ig), siRNA molecules consisting of any one of thenucleic acid sequences of SEQ ID NO: 38-67, and anti-VISTA antibodiesdescribed herein may be used in compositions, uses, and methods for thetreatment of cancer (e.g., tumors).

Examples of cancer include but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, and leukemia. More particular examples of suchcancers include squamous cell cancer, lung cancer (including small-celllung cancer, non-small cell lung cancer, adenocarcinoma of the lung, andsquamous carcinoma of the lung), cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer (includinggastrointestinal cancer), pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; multiple myeloma and post-transplant lymphoproliferativedisorder (PTLD).

The term cancer amenable for treatment by the present invention include,but not limited to, colorectal cancer, carcinoma, lymphoma, blastoma,sarcoma, and leukemia or lymphoid malignancies. More particular examplesof such cancers include bladder, ovarian, melanoma, squamous cellcancer, lung cancer (including small-cell lung cancer, non-small celllung cancer, adenocarcinoma of the lung, and squamous carcinoma of thelung), cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer (including gastrointestinal cancer), pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidney orrenal cancer, liver cancer, prostate cancer, vulval cancer, thyroidcancer, hepatic carcinoma and various types of head and neck cancer, aswell as B-cell lymphoma (including low grade/follicular non-Hodgkin'slymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; and post-transplantlymphoproliferative disorder (PTLD), as well as abnormal vascularproliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), and Meigs' syndrome. Preferably, thecancer is selected from the group consisting of colorectal cancer,breast cancer, colorectal cancer, rectal cancer, non-small cell lungcancer, non-Hodgkins lymphoma (NHL), renal cell cancer, prostate cancer,liver cancer, pancreatic cancer, soft-tissue sarcoma, kaposi's sarcoma,carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer,mesothelioma, and multiple myeloma. The cancer may be an early advanced(including metastatic) colorectal cancer, bladder cancer, ovarian canceror melanoma. The cancer may be colorectal cancer. The cancerousconditions amenable for treatment of the invention include metastaticcancers wherein VISTA expression by myeloid derived suppressor cellssuppress antitumor responses and anti-invasive immune responses. Themethod of the present invention is particularly suitable for thetreatment of vascularized tumors.

The invention is also suitable for treating cancers in combination withchemotherapy or radiotherapy or other biologics and for enhancing theactivity thereof, i.e., in individuals wherein VISTA expression bymyeloid derived suppressor cells suppress antitumor responses and theefficacy of chemotherapy or radiotherapy or biologic efficacy. Anychemotherapeutic agent exhibiting anticancer activity can be usedaccording to the present invention. Preferably, the chemotherapeuticagent may be selected from the group consisting of alkylating agents,antimetabolites, folic acid analogs, pyrimidine analogs, purine analogsand related inhibitors, vinca alkaloids, epipodopyyllotoxins,antibiotics, L-Asparaginase, topoisomerase inhibitor, interferons,platinum coordination complexes, anthracenedione substituted urea,methyl hydrazine derivatives, adrenocortical suppressant,adrenocorticosteroides, progestins, estrogens, antiestrogen, androgens,antiandrogen, and gonadotropin-releasing hormone analog. Morepreferably, the chemotherapeutic agent may be selected from the groupconsisting of 5-fluorouracil (5-FU), leucovorin (LV), irenotecan,oxaliplatin, capecitabine, paclitaxel and doxetaxel. Two or morechemotherapeutic agents can be used in a cocktail to be administered incombination with administration of the anti-VEGF antibody. One preferredcombination chemotherapy is fluorouracil-based, comprising 5-FU and oneor more other chemotherapeutic agent(s). Suitable dosing regimens ofcombination chemotherapies are known in the art and described in, forexample, Saltz, et al. (1999) Proc ASCO 18:233a and Douillard, et al.(2000) Lancet 355: 1041-7. The biologic may be another immunepotentiators such as antibodies to PD-L1, PD-L2, CTLA-4 and PD-L1,PD-L2, CTLA-4 fusion proteins as well as cytokines, growth factorantagonists and agonists, hormones and anti-cytokine antibodies.

Allergies

The VISTA polypeptides, multimeric VISTA polypeptides, VISTA fusionproteins (e.g., VISTA-Ig), and anti-VISTA antibodies described hereinmay be used in compositions, uses, and methods for the treatment ofallergies (e.g., allergic reactions to allergens).

Examples of allergens include mite antigens and pollen antigens.

Representative allergic diseases include bronchial asthma, allergicrhinitis, atopic dermatitis, and pollen and insect allergies. Allergicdiathesis is a genetic factor that can be inherited by the children ofallergic parents. Familial allergic diseases are also called atopicdiseases, and the causative, genetically transmitted factor is atopicdiathesis. “Atopic dermatitis” is a general term for an atopic disease,especially diseases accompanied by dermatitis symptoms. Preferredexamples include allergic condition is selected from the groupconsisting of eczema, allergic rhinitis, hay fever, urticaria, and foodallergies. Allergic conditions include eczema, allergic rhinitis orcoryza, hay fever, bronchial asthma, urticaria (hives) and foodallergies, and other atopic conditions.

Inflammatory Conditions and Inflammatory Diseases

The VISTA polypeptides, multimeric VISTA polypeptides, VISTA fusionproteins (e.g., VISTA-Ig), siRNA molecules consisting of any one of thenucleic acid sequences of SEQ ID NO: 38-67, and anti-VISTA antibodiesdescribed herein may be used in compositions, uses, and methods for thetreatment of inflammatory conditions and inflammatory disease.

Inflammatory conditions and inflammatory diseases, include but are notlimited to rheumatic diseases (e.g., rheumatoid arthritis,osteoarthritis, psoriatic arthritis) spondyloarthropathies (e.g.,ankylosing spondylitis, reactive arthritis, Reiter's syndrome), crystalarthropathies (e.g., gout, pseudogout, calcium pyrophosphate depositiondisease), multiple sclerosis, Lyme disease, polymyalgia rheumatica;connective tissue diseases (e.g., systemic lupus erythematosus, systemicsclerosis, polymyositis, dermatomyositis, Sjogren's syndrome);vasculitides (e.g., polyarteritis nodosa, Wegener's granulomatosis,Churg-Strauss syndrome); inflammatory conditions including consequencesof trauma or ischaemia, sarcoidosis; vascular diseases includingatherosclerotic vascular disease, atherosclerosis, and vascularocclusive disease (e.g., atherosclerosis, ischaemic heart disease,myocardial infarction, stroke, peripheral vascular disease), andvascular stent restenosis; ocular diseases including uveitis, cornealdisease, iritis, iridocyclitis, and cataracts.

Inflammatory conditions also include, but are not limited to acidReflux/Heartburn, Acne, Acne Vulgaris, Allergies and Sensitivities,Alzheimer's Disease, Asthma, Atherosclerosis and Vascular OcclusiveDisease (e.g., Atherosclerosis, Ischaemic Heart Disease, MyocardialInfarction, Stroke, Peripheral Vascular Disease) and Vascular StentRestenosis, Autoimmune Diseases, Bronchitis, Cancer, Carditis,Cataracts, Celiac Disease, Chronic Pain, Chronic Prostatitis, Cirrhosis,Colitis, Connective Tissue Diseases (e.g., Systemic Lupus Erythematosus,Systemic Sclerosis, Polymyositis, Dermatomyositis, Sjogren's Syndrome),Corneal Disease, Crohn's Disease, Crystal Arthropathies (e.g., Gout,Pseudogout, Calcium Pyrophosphate Deposition Disease), Dementia,Dermatitis, Diabetes, Dry Eyes, Eczema, Edema, Emphysema, Fibromyalgia,Gastroenteritis, Gingivitis, Glomerulonephritis, Heart Disease,Hepatitis, High Blood Pressure, Hypersensitivities, Inflammatory BowelDiseases, Inflammatory Conditions including Consequences of Trauma orIschaemia, Insulin Resistance, Interstitial Cystitis, Iridocyclitis,Iritis, Joint Pain/Arthritis/Rheumatoid Arthritis, Lyme Disease,Metabolic Syndrome (Syndrome X), Multiple Sclerosis, Myositis,Nephritis, Obesity, Ocular Diseases including Uveitis, Osteopenia,Osteoporosis, Parkinson's Disease, Pelvic Inflammatory Disease,Periodontal Disease, Polyarteritis, Polychondritis, PolymyalgiaRheumatica, Psoriasis, Reperfusion Injury, Rheumatic Arthritis,Rheumatic Diseases (e.g., Rheumatoid Arthritis, Osteoarthritis,Psoriatic Arthritis), Rheumatoid Arthritis, Sarcoidosis, Scleroderma,Sinusitis, Sjögren's Syndrome, Spastic Colon, Spondyloarthropathies(e.g., Ankylosing Spondylitis, Reactive Arthritis, Reiter's Syndrome),Systemic Candidiasis, Tendonitis, Transplant Rejection, UTI's,Vaginitis, Vascular Diseases including Atherosclerotic Vascular Disease,Vasculitides (e.g., Polyarteritis Nodosa, Wegener's Granulomatosis,Churg-Strauss Syndrome), and Vasculitis.

Graft Versus Host Disease

The VISTA polypeptides, multimeric VISTA polypeptides, VISTA fusionproteins (e.g., VISTA-Ig), siRNA molecules consisting of any one of thenucleic acid sequences of SEQ ID NO: 38-67, and anti-VISTA antibodiesdescribed herein may be used in compositions, uses, and methods for thetreatment of graft-versus-host disease (GVHD).

The invention also provides a method of treaintgraft-versus-host-disease (GVHD) comprising administration of aneffective amount of a VISTA fusion protein, optionally a VISTA-Ig fusionprotein, or the multimeric VISTA protein. A method for treatinggraft-versus-host disease (GVHD), acute graft-versus-host disease,chronic graft-versus-host disease, acute graft-versus-host diseaseassociated with stem cell transplant, chronic graft-versus-host diseaseassociated with stem cell transplant, acute graft-versus-host diseaseassociated with bone marrow transplant, acute graft-versus-host diseaseassociated with allogeneic hemapoetic stem cell transplant (HSCT), orchronic graft-versus-host disease associated with bone marrow transplantmay comprise administering of an effective amount of a VISTA fusionprotein, optionally a VISTA-Ig fusion protein, or the multimeric VISTAprotein.

The graft-versus-host disease (GVHD) may be graft-versus-host disease(GVHD), acute graft-versus-host disease, chronic graft-versus-hostdisease, acute graft-versus-host disease associated with stem celltransplant, chronic graft-versus-host disease associated with stem celltransplant, acute graft-versus-host disease associated with bone marrowtransplant, acute graft-versus-host disease associated with allogeneichemapoetic stem cell transplant (HSCT), or chronic graft-versus-hostdisease associated with bone marrow transplant. The patient treated tobe treated may have at least one symptom of graft-versus-host disease(GVHD), optionally wherein the patient exhibits acute GVHD includes butis not limited to abdominal pain, abdominal cramps, diarrhea, fever,jaundice, skin rash, vomiting, and weight loss. The patient may have atleast one symptom of chronic graft-versus-host disease (GVHD) includesbut is not limited to dry eyes, dry mouth, hair loss, hepatisis, lungdisorder, gastrointestinal tract disorders, skin rash, and skinthickening. The patient may have or may be to receive allogeneic stemcell or bone marrow transplant. The patient may have or may be toreceive autologous stem cell or bone marrow transplant.

Diagnostic Methods

The anti-VISTA and anti-VISTA conjugate antibodies which selectivelybind the VISTA and VISTA conjugate, siRNA molecules consisting of anyone of the nucleic acid sequences of SEQ ID NO: 38-67, andantigen-binding fragments thereof, may be used in diagnostic methods fordetecting the presence or absence of an VISTA and VISTA conjugate.Anti-VISTA and anti-VISTA conjugate antibodies may be used in methodscomprising (a) contacting a test sample with an antibody, or fragmentthereof, that binds a VISTA or VISTA conjugate, and (b) assaying forantibody-epitope complexes. The antibody-epitope complex may be detectedby Western blot, radioimmunoassay, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassay, immunoprecipitation assay, precipitationreaction, gel diffusion precipitation reaction, immunodiffusion assay,agglutination assay, complement-fixation assay, immunohistochemicalassay, fluorescent immunoassay, and protein A immunoassay. The samplemay be sample is a tissue biopsy, lymph, urine, cerebrospinal fluid,amniotic fluid, inflammatory exudate, blood, serum, stool, or liquidcollected from the colorectal tract.

The antibodies which selectively bind a VISTA and VISTA conjugate may berecombinant. The fragments of antibodies which selectively bind a VISTAand VISTA conjugate may be a Fab, Fab′, F(ab′)2, Fv, CDR, paratope, orportion of an antibody that is capable of binding the antigen. Theantibodies which selectively bind a VISTA and VISTA conjugate may bechimeric, humanized, anti-idiotypic, single-chain, bifunctional, orco-specific. The antibodies which selectively bind a VISTA and VISTAconjugate may be or fragment is conjugated to a label, including but notlimited to a chemiluminescent label, paramagnetic label (e.g., aluminum,manganese, platinum, oxygen, lanthanum, lutetium, scandium, yttrium, orgallium), an MRI contrast agent, fluorescent label, bioluminescentlabel, or radioactive label.

Additionally, VISTA and VISTA conjugate, antibody which selectively binda VISTA and VISTA conjugate, and antigen-binding fragments thereof, maybe attached to a solid support (e.g., bead, test tube, sheet, culturedish, or test strip) such as an array.

The method may comprise imaging a VISTA polypeptide or VISTA conjugateby positron emission tomography (PET), CCD low-light monitoring system,x-ray, CT scanning, scintigraphy, photo acoustic imaging, single photonemission computed tomography (SPECT), magnetic resonance imaging (MRI),ultrasound, paramagnetic imaging, and endoscopic optical coherencetomography.

Screening Assays

The invention provides a method for identifying modulators (“screeningassay”), i.e., candidate or test compounds or agents (e.g., peptides,peptidomimetics, small molecules or other drugs) which bind to VISTApolypeptides, have a stimulatory or inhibitory effect on, for example,VISTA expression or VISTA activity, or have a stimulatory or inhibitoryeffect on the interaction between VISTA and its natural bindingpartner(s).

Assays for screening candidate or test compounds which bind to the VISTApolypeptide or biologically active portion thereof, e.g., modulate theability of the VISTA polypeptide to interact with its natural bindingpartner(s) may comprise contacting a candidate compound with a VISTApolypeptide and testing for the modulating of the ability of the VISTApolypeptide to interact with its natural binding partner. Assays forscreening candidate or test compounds which bind to or modulate theactivity of a VISTA protein or polypeptide or biologically activeportion thereof may comprise contacting a VISTA polypeptide and testingfor binding between the VISTA polypeptide and the candidate agent.Assays for screening candidate or test compounds which have astimulatory or inhibitory effect on immune functions negativelyregulated by VISTA such as are identified herein or based on its effecton the interaction of between VISTA and its natural binding partner(s).These VISTA related functions include by way of example inhibitingcytokine production (e.g., Il-2, gamma interferon by T cells,suppressing moderate CD28 costimulation, inhibiting CD4+ and CD8+ T cellproliferation, suppressing proliferation of naïve and memory CD4+ Tcells, and suppressing TCR activation without inducing apoptosis.) Thetest compounds of the present invention can be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the “one-bead one-compound”library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds. Lam (1997) Anticancer Drug Des. 12:145.

An assay may be a cell-based assay in which a cell which expresses aVISTA polypeptide or biologically active portion thereof comprisingcontacting a VISTA polypeptide or biologically active portion thereofwith a test compound, and determining the ability of the test compoundto modulate VISTA activity. Determining the ability of the test compoundto modulate VISTA activity can be accomplished by monitoring, forexample, the ability of VISTA to bind to its natural binding partner(s),and modulate immune cell activity. The immune cell can be a T cell, a Bcell, or a myeloid cell. Determining the ability of the test compound tomodulate VISTA binding to its counter-receptor can be accomplished, forexample, by coupling VISTA with a radioisotope or enzymatic label tomonitor the ability of a test compound to modulate VISTA binding to Tcells which express the VISTA counter-receptor. Determining the abilityof the test compound to bind VISTA can be accomplished, for example, bycoupling the compound with a radioisotope or enzymatic label such thatbinding of the compound to VISTA can be determined by detecting thelabeled VISTA compound in a complex.

Assays may be used to determine the ability of a compound to interactwith VISTA without the labeling of any of the interactants. For example,a microphysiometer can be used to detect the interaction of a compoundwith VISTA without the labeling of either the compound or the VISTA.McConnell, H. M. et al. (1992) Science 257:1906-1912. A microphysiometer(e.g., Cytosensor) is an analytical instrument that measures the rate atwhich a cell acidities its environment using a light-addressablepotentiometric sensor (LAPS). Changes in this acidification rate can beused as an indicator of the interaction between a compound and VISTA.

An assay may be a cell-based assay comprising contacting a T cellexpressing a VISTA binding partner with a test compound and determiningthe ability of the test compound to modulate (e.g., stimulate orinhibit) the activity of the VISTA binding partner. Determining theability of the test compound to modulate the activity of a VISTA bindingpartner can be accomplished, for example, by determining the ability ofthe VISTA polypeptide to bind to or interact with the VISTA bindingpartner.

Determining the ability of the VISTA polypeptide, or a biologicallyactive fragment thereof, to bind to or interact with a VISTA bindingpartner, can be accomplished by one of the methods described above fordetermining direct binding. In an embodiment, determining the ability ofthe VISTA polypeptide to bind to or interact with a VISTA bindingpartner can be accomplished by determining the activity of the bindingpartner. For example, the activity of the binding partner can bedetermined by detecting induction of a cellular second messenger (e.g.,tyrosine kinase or phosphatase activity), detecting catalytic/enzymaticactivity of an appropriate substrate, detecting the induction of areporter gene (comprising a target-responsive regulatory elementoperatively linked to a nucleic acid encoding a detectable marker, e.g.,luciferase), or detecting a target-regulated cellular response. Forexample, determining the ability of the VISTA polypeptide to bind to orinteract with a natural VISTA binding partner, can be accomplished bymeasuring the ability of a compound to modulate immune cellcostimulation or inhibition in a proliferation assay, or by interferingwith the ability of a VISTA polypeptide to bind to antibodies thatrecognize a portion of the VISTA polypeptide. In one embodiment,compounds that modulate T cell activation can be identified bydetermining the ability of a compound to modulate T cell proliferationor cytokine production. In an embodiment, compounds that modulate T cellactivation can be identified by determining the ability of a compound tomodulate T cell proliferation or cytokine production at more than oneantigen concentration.

An assay may be a cell-free assay in which a VISTA polypeptide orbiologically active portion thereof is contacted with a test compoundand the ability of the test compound to bind to the VISTA polypeptide orbiologically active portion thereof is determined. Preferredbiologically active portions of the VISTA polypeptides to be used inassays of the present invention include fragments which participate ininteractions with non-VISTA molecules, e.g., at least a portion of anextracellular domain which binds to a VISTA binding partner. Binding ofthe test compound to the VISTA polypeptide can be determined eitherdirectly or indirectly as described above.

The assay may be a cell-free assay in which a VISTA polypeptide orbiologically active portion thereof is contacted with a test compoundand the ability of the test compound to modulate (e.g., stimulate orinhibit) the activity of the VISTA polypeptide or biologically activeportion thereof is determined Determining the ability of the testcompound to modulate the activity of a VISTA polypeptide can beaccomplished, for example, by determining the ability of the VISTApolypeptide to bind to a VISTA binding partner by one of the methodsdescribed above for determining direct binding. The cell-free assays ofthe present invention are amenable to use of both soluble and/ormembrane-bound forms of polypeptides (e.g., VISTA polypeptides orbiologically active portions thereof, or binding partners to which VISTAbinds). In the case of cell-free assays in which a membrane-bound form apolypeptide is used (e.g., a cell-surface VISTA), it may be desirable toutilize a solubilizing agent such that the membrane-bound form of thepolypeptide is maintained in solution. Examples of such solubilizingagents include non-ionic detergents such as n-octylglucoside,n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit,Isotridecypoly(ethylene glycol ether)n,3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl.dbd.N,N-dimethyl-3-ammonio-1-propane sulfonate.

In assay methods, it may be desirable to immobilize either VISTA or itsbinding partner to facilitate separation of complexed from uncomplexedforms of one or both of the polypeptides, as well as to accommodateautomation of the assay. Binding of a test compound to a VISTApolypeptide, or interaction of a VISTA polypeptide with its bindingpartner in the presence and absence of a candidate compound, can beaccomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtitre plates, test tubes, andmicro-centrifuge tubes. In one embodiment, a fusion protein can beprovided which adds a domain that allows one or both of the polypeptidesto be bound to a matrix. For example, glutathione-S-transferase/VISTAfusion proteins or glutathione-S-transferase/binding partner fusionproteins can be adsorbed onto glutathione SEPHAROSE® beads (SigmaChemical, St. Louis, Mo.) or glutathione derivatized microtitre plates,which are then combined with the test compound or the test compound andeither the non-adsorbed binding partner polypeptide or VISTApolypeptide, and the mixture incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads or microtitre plate wells are washed toremove any unbound components, the matrix is immobilized in the case ofbeads, and complex formation is determined either directly orindirectly, for example, as described above. Alternatively, thecomplexes can be dissociated from the matrix, and the level of VISTAbinding or activity determined using standard techniques. Othertechniques for immobilizing polypeptides on matrices can also be used inthe screening assays of the invention. Determining the ability of thetest compound to modulate the activity of a VISTA polypeptide may beaccomplished by determining the ability of the test compound to modulatethe activity of a molecule that functions downstream of VISTA, e.g., byinteracting with the cytoplasmic domain of a VISTA binding partner. Forexample, levels of second messengers, the activity of the interactingmolecule on an appropriate target, or the binding of the interactor toan appropriate target can be determined as previously described.

Modulators of VISTA expression may be identified in a method wherein acell is contacted with a candidate compound and the expression of VISTAmRNA or polypeptide in the cell is determined. The level of expressionof VISTA mRNA or polypeptide in the presence of the candidate compoundis compared to the level of expression of VISTA mRNA or polypeptide inthe absence of the candidate compound. The candidate compound can thenbe identified as a modulator of VISTA expression based on thiscomparison if the change is statistically significant.

The VISTA polypeptides may be used as “bait proteins” in a two-hybridassay or three-hybrid assay (See, e.g., U.S. Pat. No. 5,283,317; Zervos,et al. (1993) Cell 72:223-232; Madura, et al. (1993) J. Biol. Chem.268:12046-12054; Bartel, et al. (1993) Biotechniques 14:920-924;Iwabuchi, et al. (1993) Oncogene 8:1693-1696; and WO 94/10300), toidentify other polypeptides which bind to or interact with VISTA(“VISTA-binding proteins”, “VISTA binding partners”, or “VISTA-bp”) andare involved in VISTA activity. Such VISTA-binding proteins are alsolikely to be involved in the propagation of signals by the VISTApolypeptides or VISTA targets as, for example, downstream elements of aVISTA-mediated signaling pathway. Alternatively, such VISTA-bindingpolypeptides may be VISTA inhibitors. The two-hybrid system is based onthe modular nature of most transcription factors, which consist ofseparable DNA-binding and activation domains. Briefly, the assayutilizes two different DNA constructs. In one construct, the gene thatcodes for a VISTA polypeptide is fused to a gene encoding the DNAbinding domain of a known transcription factor (e.g, GAL-4). In theother construct, a DNA sequence, from a library of DNA sequences, thatencodes an unidentified polypeptide (“prey” or “sample”) is fused to agene that codes for the activation domain of the known transcriptionfactor. If the “bait” and the “prey” polypeptides are able to interact,in vivo, forming a VISTA-dependent complex, the DNA-binding andactivation domains of the transcription factor are brought into closeproximity. This proximity allows transcription of a reporter gene (e.g,LacZ) which is operably linked to a transcriptional regulatory siteresponsive to the transcription factor. Expression of the reporter genecan be detected and cell colonies containing the functionaltranscription factor can be isolated and used to obtain the cloned genewhich encodes the polypeptide which interacts with the VISTApolypeptide.

A combination of two or more of the assays described herein. Forexample, a modulating agent may be identified using a cell-based or acell-free assay, and the ability of the agent to modulate the activityof a VISTA polypeptide can be confirmed in vivo, e.g., in an animal suchas an animal model for cellular transformation and/or tumorigenesis.

This invention further pertains to novel agents identified by theabove-described screening assays. An agent as identified in the methodsdescribed herein in an appropriate animal model. For example, an agentidentified as described herein (e.g., a VISTA modulating agent, anantisense VISTA nucleic acid molecule, a VISTA-specific antibody, or aVISTA binding partner) can be used in an animal model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal model to determine the mechanism of action of such an agent.Furthermore, this invention pertains to uses of novel agents identifiedby the above-described screening assays for treatments as describedherein.

Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) map their respective genes on a chromosome; and, thus, locate generegions associated with genetic disease; (ii) identify an individualfrom a minute biological sample (tissue typing); and (iii) aid inforensic identification of a biological sample. These applications aredescribed in the subsections below.

Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has beenisolated, this sequence can be used to map the location of the gene on achromosome. This process is called chromosome mapping. Accordingly,portions or fragments of the VISTA nucleotide sequences, describedherein, can be used to map the location of the VISTA genes on achromosome. The mapping of the VISTA sequences to chromosomes is animportant first step in correlating these sequences with genesassociated with disease. Briefly, VISTA genes can be mapped tochromosomes by preparing PCR primers (preferably 15-25 bp in length)from the VISTA nucleotide sequences. Computer analysis of the VISTAsequences can be used to predict primers that do not span more than oneexon in the genomic DNA, thus complicating the amplification process.These primers can then be used for PCR screening of somatic cell hybridscontaining individual human chromosomes. Only those hybrids containingthe human gene corresponding to the VISTA sequences will yield anamplified fragment. Somatic cell hybrids are prepared by fusing somaticcells from different mammals (e.g., human and mouse cells). As hybridsof human and mouse cells grow and divide, they gradually lose humanchromosomes in random order, but retain the mouse chromosomes. By usingmedia in which mouse cells cannot grow, because they lack a particularenzyme, but human cells can, the one human chromosome that contains thegene encoding the needed enzyme will be retained. By using variousmedia, panels of hybrid cell lines can be established. Each cell line ina panel contains either a single human chromosome or a small number ofhuman chromosomes, and a full set of mouse chromosomes, allowing easymapping of individual genes to specific human chromosomes. D'Eustachio,et al. (1983) Science 220: 919-924. Somatic cell hybrids containing onlyfragments of human chromosomes can also be produced by using humanchromosomes with translocations and deletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermal cycler. Using the VISTAnucleotide sequences to design oligonucleotide primers, sublocalizationcan be achieved with panels of fragments from specific chromosomes.Other mapping strategies which can similarly be used to map a VISTAsequence to its chromosome include in situ hybridization (described inFan, et al. (1990) Proc Natl. Acad. Sci. USA 87:6223-27), pre-screeningwith labeled flow-sorted chromosomes, and pre-selection by hybridizationto chromosome specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical such ascolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosome, so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases willsuffice to get good results in a reasonable amount of time. For a reviewof this technique, see Verma, et al. Human Chromosomes: A Manual ofbasic Techniques (Pergamon Press, New York 1988). Reagents forchromosome mapping can be used individually to mark a single chromosomeor a single site on that chromosome, or panels of reagents can be usedfor marking multiple sites and/or multiple chromosomes. Reagentscorresponding to noncoding regions of the genes actually are preferredfor mapping purposes. Coding sequences are more likely to be conservedwithin gene families, thus increasing the chance of cross hybridizationduring chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data Ultimately, complete sequencing of genes fromseveral individuals can be performed to confirm the presence of amutation and to distinguish mutations from polymorphisms.

Tissue Typing

The VISTA sequences of the present invention can also be used toidentify individuals from minute biological samples. Furthermore, thesequences of the present invention can be used to provide an alternativetechnique which determines the actual base-by-base DNA sequence ofselected portions of an individual's genome. Thus, the VISTA nucleotidesequences described herein can be used to prepare two PCR primers fromthe 5′ and 3′ ends of the sequences. These primers can then be used toamplify an individual's DNA and subsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The VISTA nucleotide sequences of the invention uniquely representportions of the human genome. Allelic variation occurs to some degree inthe coding regions of these sequences, and to a greater degree in thenoncoding regions. It is estimated that allelic variation betweenindividual humans occurs with a frequency of about once per each 500bases. Each of the sequences described herein can, to some degree, beused as a standard against which DNA from an individual can be comparedfor identification purposes. Because greater numbers of polymorphismsoccur in the noncoding regions, fewer sequences are necessary todifferentiate individuals. The noncoding sequences of SEQ ID NO: 1 or 4can comfortably provide positive individual identification with a panelof perhaps 10 to 1,000 primers which each yield a noncoding amplifiedsequence of 100 bases. If predicted coding sequences, such as those inSEQ ID NO: 3 or 6 are used, a more appropriate number of primers forpositive individual identification would be 500-2000.

If a panel of reagents from VISTA nucleotide sequences described hereinis used to generate a unique identification database for an individual,those same reagents can later be used to identify tissue from thatindividual. Using the unique identification database, positiveidentification of the individual, living or dead, can be made fromextremely small tissue samples.

Use of VISTA Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e., another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NO: 1 or 3 are particularlyappropriate for this use as greater numbers of polymorphisms occur inthe noncoding regions, making it easier to differentiate individualsusing this technique. Examples of polynucleotide reagents include theVISTA nucleotide sequences or portions thereof, e.g., fragments derivedfrom the noncoding regions of SEQ ID NO: 1 or 3 having a length of atleast 20 bases, preferably at least 30 bases. The VISTA nucleotidesequences described herein can further be used to provide polynucleotidereagents, e.g., labeled or labelable probes which can be used in, forexample, an in situ hybridization technique, to identify a specifictissue, e.g., lymphocytes. This can be very useful in cases where aforensic pathologist is presented with a tissue of unknown origin.Panels of such VISTA probes can be used to identify tissue by speciesand/or by organ type. In a similar fashion, these reagents, e.g., VISTAprimers or probes can be used to screen tissue culture for contamination(i.e., screen for the presence of a mixture of different types of cellsin a culture).

Diagnostic Assays

An exemplary method for detecting the presence or absence of VISTApolypeptide or nucleic acid in a biological sample involves obtaining abiological sample from a test subject and contacting the biologicalsample with a compound or an agent capable of detecting VISTApolypeptide or nucleic acid (e.g., mRNA or genomic DNA) that encodesVISTA polypeptide such that the presence of VISTA polypeptide or nucleicacid is detected in the biological sample. A preferred agent fordetecting VISTA mRNA or genomic DNA is a labeled nucleic acid probecapable of hybridizing to VISTA mRNA or genomic DNA. The nucleic acidprobe can be, for example, the VISTA nucleic acid set forth in SEQ IDNO: 1 or 3, or a portion thereof, such as an oligonucleotide of at least15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient tospecifically hybridize under stringent conditions to VISTA mRNA orgenomic DNA. Other suitable probes for use in the diagnostic assays ofthe invention are described herein. A preferred agent for detectingVISTA polypeptide is an antibody capable of binding to VISTApolypeptide, preferably an antibody with a detectable label. Antibodiescan be polyclonal, or more preferably, monoclonal. An intact antibody,or a fragment thereof (e.g., Fab or F(ab′)₂) can be used. The term“labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin. The term“biological sample” is intended to include tissues, cells, andbiological fluids isolated from a subject, as well as tissues, cells,and fluids present within a subject. That is, the detection method ofthe invention can be used to detect VISTA mRNA, polypeptide, or genomicDNA in a biological sample in vitro as well as in vivo. For example, invitro techniques for detection of PD-L2 mRNA include Northernhybridizations and in situ hybridizations. in vitro techniques fordetection of VISTA polypeptide include enzyme linked immunosorbentassays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence. in vitro techniques for detection of VISTA genomicDNA include Southern hybridizations. Furthermore, in vivo techniques fordetection of VISTA polypeptide include introducing into a subject alabeled anti-VISTA antibody. For example, the antibody can be labeledwith a radioactive marker whose presence and location in a subject canbe detected by standard imaging techniques. In one embodiment, thebiological sample contains polypeptide molecules from the test subject.Alternatively, the biological sample can contain mRNA molecules from thetest subject or genomic DNA molecules from the test subject. A preferredbiological sample is a serum sample isolated by conventional means froma subject. In another embodiment, the methods further involve obtaininga control biological sample from a control subject, contacting thecontrol sample with a compound or agent capable of detecting VISTApolypeptide, mRNA, or genomic DNA, such that the presence of VISTApolypeptide, mRNA or genomic DNA is detected in the biological sample,and comparing the presence of VISTA polypeptide, mRNA or genomic DNA inthe control sample with the presence of VISTA polypeptide, mRNA orgenomic DNA in the test sample.

The invention also encompasses kits for detecting the presence of VISTAin a biological sample. For example, the kit can comprise a labeledcompound or agent capable of detecting VISTA polypeptide or mRNA in abiological sample; means for determining the amount of VISTA in thesample; and means for comparing the amount of VISTA in the sample with astandard. The compound or agent can be packaged in a suitable container.The kit can further comprise instructions for using the kit to detectVISTA polypeptide or nucleic acid.

Prognostic Assays

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant or unwanted VISTA expression or activity. Asused herein, the term “aberrant” includes a VISTA expression or activitywhich deviates from the wild type VISTA expression or activity. Aberrantexpression or activity includes increased or decreased expression oractivity, as well as expression or activity which does not follow thewild type developmental pattern of expression or the subcellular patternof expression. For example, aberrant VISTA expression or activity isintended to include the cases in which a mutation in the VISTA genecauses the VISTA gene to be under-expressed or over-expressed andsituations in which such mutations result in a non-functional VISTApolypeptide or a polypeptide which does not function in a wild-typefashion, e.g., a polypeptide which does not interact with a VISTAbinding partner, or one which interacts with a non-VISTA bindingpartner. As used herein, the term “unwanted” includes an unwantedphenomenon involved in a biological response such as immune cellactivation. For example, the term unwanted includes a VISTA expressionor activity which is undesirable in a subject.

The assays described herein, such as the preceding diagnostic assays orthe following assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with a misregulation in VISTApolypeptide activity or nucleic acid expression, such as an autoimmunedisorder, an immunodeficiency disorder, an immune system disorder suchas autoimmunity, allergic or inflammatory disorder or cancer. Thus, thepresent invention provides a method for identifying a disease ordisorder associated with aberrant or unwanted VISTA expression oractivity in which a test sample is obtained from a subject and VISTApolypeptide or nucleic acid (e.g., mRNA or genomic DNA) is detected,wherein the presence of VISTA polypeptide or nucleic acid is diagnosticfor a subject having or at risk of developing a disease or disorderassociated with aberrant or unwanted VISTA expression or activity. Asused herein, a “test sample” refers to a biological sample obtained froma subject of interest. For example, a test sample can be a biologicalfluid (e.g., cerebrospinal fluid or serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant or unwanted VISTA expression or activity. Forexample, such methods can be used to determine whether a subject can beeffectively treated with an agent for an autoimmune disorder,immunodeficiency disorder, immune system cancer, or allergic orinflammatory disorder. Thus, the present invention provides methods fordetermining whether a subject can be effectively treated with an agentfor a disorder associated with aberrant or unwanted VISTA expression oractivity in which a test sample is obtained and VISTA polypeptide ornucleic acid expression or activity is detected (e.g., wherein theabundance of VISTA polypeptide or nucleic acid expression or activity isdiagnostic for a subject that can be administered the agent to treat adisorder associated with aberrant or unwanted VISTA expression oractivity). The methods of the invention can also be used to detectgenetic alterations in a VISTA gene, thereby determining if a subjectwith the altered gene is at risk for a disorder characterized bymisregulation in VISTA polypeptide activity or nucleic acid expression,such as an autoimmune disorder, an immunodeficiency disorder, an immunesystem cancer, an allergic disorder, or an inflammatory disorder. Themethods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a VISTA gene.Furthermore, any cell type or tissue in which VISTA is expressed may beutilized in the prognostic assays described herein.

Immunoassays

The VISTA and VISTA conjugate, antibodies and antigen-binding fragmentsthat bind the VISTA and VISTA conjugate, may be used in immunoassays toqualitatively or quantitatively detect and analyze markers in a sample.This method comprises providing an antibody specifically binds to aVISTA or VISTA conjugate; contacting a sample with the antibody; anddetecting the presence of a complex of the antibody bound to the markerin the sample.

VISTA and VISTA conjugate may be detected and/or quantified using any ofa number of well recognized immunological binding assays. Useful assaysinclude, for example, an enzyme immune assay (EIA) such as enzyme-linkedimmunosorbent assay (ELISA), a radioimmunoassay (RIA), a Western blotassay, or a slot blot assay. See, e.g., U.S. Pat. Nos. 4,366,241;4,376,110; 4,517,288; and 4,837,168. Generally, a sample obtained from asubject can be contacted with the antibody specifically binds the VISTAor VISTA conjugate.

Optionally, the antibody can be fixed to a solid support to facilitatewashing and subsequent isolation of the complex, prior to contacting theantibody with a sample. Examples of solid supports include but are notlimited to glass or plastic in the form of, e.g., a microtiter plate, astick, a bead, or a microbead. Antibodies may be attached to a solidsupport.

After incubating the sample with antibodies, the mixture is washed andthe antibody-marker complex formed may be detected. This can beaccomplished by incubating the washed mixture with a detection reagent.Alternatively, the marker in the sample can be detected using anindirect assay, wherein, for example, a second, labeled antibody is usedto detect bound marker-specific antibody, and/or in a competition orinhibition assay wherein, for example, a monoclonal antibody which bindsto a distinct epitope of the marker are incubated simultaneously withthe mixture.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,marker, volume of solution, concentrations. Usually the assays will becarried out at ambient temperature, although they can be conducted overa range of temperatures (e.g., 10° C.-40° C.).

The immunoassay can be used to determine a test amount of a marker in asample from a subject. First, a test amount of a marker in a sample maybe detected using the immunoassay methods described above. If a markeris present in the sample, it will form an antibody-marker complex withan antibody specifically binds the marker under suitable incubationconditions described above. The amount of an antibody-marker complex canoptionally be determined by comparing to a standard. As noted above, thetest amount of marker need not be measured in absolute units, as long asthe unit of measurement can be compared to a control amount and/orsignal. Several immunoassays are known in the art and the VISTApolypeptide or VISTA conjugate described herein may used in suchimmunoassays including but not limited to radio-immunoassay (RIA),enzyme linked immunosorbent assay (ELISA), magnetic immunoassay,immunoblot, Western blot, immunoprecipitation assays,immunohistochemical analysis, and fluorescence activated cell sorting(FACS). See Wild, (2008) [Ed.] The Immunoassay Handbook [3^(rd) Ed.]Elsevier.

Radio-Imaging Methods

The VISTA and VISTA conjugate may be used in radio-imaging methods todiagnosis cancer including pancreatic and colorectal cancer, or monitorthe progression of tumors. These methods include but are not limited to,positron emission tomography (PET) single photon emission computedtomography (SPECT). Both of these techniques are non-invasive, and canbe used to detect and/or measure a wide variety of tissue events and/orfunctions, such as detecting cancerous cells for example. SPECT mayoptionally be used with two labels simultaneously. See U.S. Pat. No.6,696,686.

Commercial Applications and Methods

The present invention further provides for the production of VISTA andVISTA conjugate to reach commercial quantities. The VISTA and VISTAconjugate may be produced on a large scale, stored if necessary, andsupplied to hospitals, clinicians or other healthcare facilities.

Methods of production, storage, and distribution of VISTA and VISTAconjugate may be produced by the methods disclosed herein. Followingproduction, the VISTA and VISTA conjugate may be harvested, purified,and optionally stored prior to a patient's treatment. For example, oncea patient presents with an indication such as, for example, cancer,autoimmune disease, or inflammatory condition, VISTA and VISTA conjugatemay be ordered and provided in a timely manner. Accordingly, the presentinvention relates to methods of producing VISTA and VISTA conjugate toattain antibodies on a commercial scale, pharmaceutical compositionscomprising antibodies and antigen binding fragments thereof whichselectively bind to VISTA and VISTA conjugate, as well as methods ofproviding (i.e., producing, optionally storing, and selling) the VISTAand VISTA conjugate to hospitals and clinicians. The production of VISTAand VISTA conjugate may be scaled up for commercial use.

The present invention also provides for methods of conducting apharmaceutical business comprising establishing a distribution systemfor distributing the preparation for sale or may include establishing asales group for marketing the pharmaceutical preparation.

Library of Nucleic Acids

A variegated library of VISTA (PD-L3) variants may be generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of VISTA (PD-L3) variantsmay be produced by, for example, enzymatically ligating a mixture ofsynthetic oligonucleotides into gene sequences such that a degenerateset of potential VISTA (PD-L3) sequences expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display) containing the set of VISTA (PD-L3) sequencestherein. There are a variety of methods which can be used to producelibraries of potential VISTA (PD-L3) variants from a degenerateoligonucleotide sequence. Chemical synthesis of a degenerate genesequence can be performed in an automatic DNA synthesizer, and thesynthetic gene then ligated into an appropriate expression vector. Useof a degenerate set of genes allows for the provision, in one mixture,of all of the sequences encoding the desired set of potential VISTA(PD-L3) sequences. Methods for synthesizing degenerate oligonucleotidesare known in the art. See, e.g., Narang (1983) Tetrahedron 39:3;Itakura, et al. (1984) Annu. Rev. Biochem. 53:323; Itakura, et al.(1984) Science 198:1056; Ike, et al. (1983) Nucleic Acids Res. 11:477.

In addition, libraries of fragments of a VISTA (PD-L3) polypeptidecoding sequence may be used to generate a variegated population of VISTA(PD-L3) fragments for screening and subsequent selection of variants ofa VISTA (PD-L3) polypeptide. A library of coding sequence fragments canbe generated by treating a double stranded PCR fragment of a VISTA(PD-L3) coding sequence with a nuclease under conditions wherein nickingoccurs only about once per molecule, denaturing the double stranded DNA,renaturing the DNA to form double stranded DNA which can includesense/antisense pairs from different nicked products, removing singlestranded portions from reformed duplexes by treatment with 51 nuclease,and ligating the resulting fragment library into an expression vector.By this method, an expression library can be derived which encodesN-terminal, C-terminal and internal fragments of various sizes of theVISTA (PD-L3) polypeptide.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of VISTA (PD-L3)polypeptides. The most widely used techniques, which are amenable tohigh through-put analysis, for screening large gene libraries typicallyinclude cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates isolation of the vectorencoding the gene whose product was detected. Recursive ensemblemutagenesis (REM), a new technique which enhances the frequency offunctional mutants in the libraries, can be used in combination with thescreening assays to identify VISTA (PD-L3) variants. Arkin and Youvan(1992) Proc Natl. Acad. Sci. USA 89:7811-7815; Delagrave et al. (1993)Protein Eng. 6(3):327-331.

Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, and monitoring clinicaltrials are used for prognostic (predictive) purposes to thereby treat anindividual prophylactically. Accordingly, one aspect of the presentinvention relates to diagnostic assays for determining VISTA polypeptideand/or nucleic acid expression as well as VISTA activity, in the contextof a biological sample (e.g., blood, serum, cells, or tissue) to therebydetermine whether an individual is afflicted with a disease or disorder,or is at risk of developing a disorder, associated with aberrant orunwanted VISTA expression or activity. The invention also provides forprognostic (or predictive) assays for determining whether an individualis at risk of developing a disorder associated with VISTA polypeptide,nucleic acid expression or activity. For example, mutations in a VISTAgene can be assayed in a biological sample. Such assays can be used forprognostic or predictive purpose to thereby prophylactically treat anindividual prior to the onset of a disorder characterized by orassociated with VISTA polypeptide, nucleic acid expression or activity.

Another embodiment of the invention pertains to monitoring the influenceof agents (e.g., drugs, compounds) on the expression or activity ofVISTA in clinical trials. These and other agents are described infurther detail in the following sections.

Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of a VISTA polypeptide (e.g., the modulation of cellproliferation and/or migration) can be applied not only in basic drugscreening, but also in clinical trials. For example, the effectivenessof an agent determined by a screening assay as described herein toincrease VISTA gene expression, polypeptide levels, or upregulate VISTAactivity, can be monitored in clinical trials of subjects exhibitingdecreased VISTA gene expression, polypeptide levels, or downregulatedVISTA activity. Alternatively, the effectiveness of an agent determinedby a screening assay to decrease VISTA gene expression, polypeptidelevels, or downregulate VISTA activity, can be monitored in clinicaltrials of subjects exhibiting increased VISTA gene expression,polypeptide levels, or VISTA activity. As noted VISTA is expressed onmany hematopoietic cell types including APCs (macrophages and myeloiddendritic cells), and CD4+ T cells, and more specifically is expressedon CD11c⁺ DCs, CD4⁺ T cells (including both Foxp3⁻ effector T cells andFoxp3⁺ nTregs), CD8⁺ T cells, and Gr1⁺ granulocytes, and expressed atlow levels on B cells and NK cells In such clinical trials, theexpression or activity of a VISTA gene, and preferably, other genes thathave been implicated in, for example, a VISTA-associated disorder can beused as a “read out” or marker of the phenotype of a particular cell.

For example, and not by way of limitation, genes, including VISTA, thatare modulated in cells by treatment with an agent (e.g., compound, drugor small molecule) which modulates VISTA activity (e.g., identified in ascreening assay as described herein) can be identified. Thus, to studythe effect of agents on VISTA-associated disorders, for example, in aclinical trial, cells can be isolated and RNA prepared and analyzed forthe levels of expression of VISTA and other genes implicated in theVISTA-associated disorder, respectively. The levels of gene expression(e.g., a gene expression pattern) can be quantified by Northern blotanalysis or RT-PCR, as described herein, or alternatively by measuringthe amount of polypeptide produced, by one of the methods as describedherein, or by measuring the levels of activity of VISTA or other genes.In this way, the gene expression pattern can serve as a marker,indicative of the physiological response of the cells to the agent.Accordingly, this response state may be determined before, and atvarious points during treatment of the individual with the agent. In anembodiment, the present invention provides a method for monitoring theeffectiveness of treatment of a subject with an agent (e.g., an agonist,antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, smallmolecule, or other drug candidate identified by the screening assaysdescribed herein) including the steps of (i) obtaining apre-administration sample from a subject prior to administration of theagent; (ii) detecting the level of expression of a VISTA polypeptide,mRNA, or genomic DNA in the preadministration sample; (iii) obtainingone or more post-administration samples from the subject; (iv) detectingthe level of expression or activity of the VISTA polypeptide, mRNA, orgenomic DNA in the post-administration samples; (v) comparing the levelof expression or activity of the VISTA polypeptide, mRNA, or genomic DNAin the pre-administration sample with the VISTA polypeptide, mRNA, orgenomic DNA in the post administration sample or samples; and (vi)altering the administration of the agent to the subject accordingly. Forexample, increased administration of the agent may be desirable toincrease the expression or activity of VISTA to higher levels thandetected, i.e., to increase the effectiveness of the agent.Alternatively, decreased administration of the agent may be desirable todecrease expression or activity of VISTA to lower levels than detected,i.e., to decrease the effectiveness of the agent. According to such anembodiment, VISTA expression or activity may be used as an indicator ofthe effectiveness of an agent, even in the absence of an observablephenotypic response.

All publications (e.g., Non-Patent Literature), patents, patentapplication publications, and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains. All such publications (e.g.,Non-Patent Literature), patents, patent application publications, andpatent applications are herein incorporated by reference to the sameextent as if each individual publication, patent, patent applicationpublication, or patent application was specifically and individuallyindicated to be incorporated by reference.

In order that the invention herein described may be fully understood,the foregoing detailed description is set forth. Various embodiments ofthe invention are described in detail and may be further illustrated bythe provided examples.

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 Cloning and Sequence Analysis of VISTA (PD-L3)

VISTA (PD-L3) and Treg-sTNF were identified by global transcriptionalprofiling of resting Treg, Treg activated with αCD3, and Treg activatedwith αCD3/αGITR. αGITR was selected for this analysis as triggering ofGITR on Treg has been shown to extinguish their contact-dependentsuppressive activity (Shimizu, et al. (2002) supra). VISTA (PD-L3) andTreg-sTNF were identified on AFFIMETRIX® DNA arrays based on theirunique expression patterns (Table 2). VISTA (PD-L3) exhibited anincrease in expression in αCD3 activated Treg and reduced expression inthe presence of αGITR; and Treg-sTNF exhibited a αCD3/αGITR-dependentincrease in expression.

Purified CD4+CD25+ T cells were stimulated in culture overnight withnone, αCD3, or αCD3/αGITR, and RNA isolated for real-time PCR analysis.Expression listed is relative to actin.

TABLE 2 Relative Expression mRNA None αCD3 αCD3/αGITR VISTA (PD-L3) 6 107 T^(reg)-sTNF 0.2 0.3 1.5

AFFIMETRIX® analysis of activated vs. resting CD25+CD4+ nTregs revealedthe expression of a gene product (RIKEN cDNA 4632428N05, or4632428N05Rik) with unknown function but with sequence homology to theIg superfamily.

More specifically, a 930 bp gene product was cloned from the CD4+ T cellcDNA library, which matched the predicted size and sequence.Silico-sequence and structural analysis predicts a transmembrane proteinof 309 amino acids upon maturation, with an extracellular domain of 159amino acids, a transmembrane domain of 22 amino acids and a cytoplasmictail of 95 amino acids (FIG. 1A). Amino acid sequence alignment revealsan extracellular Immunoglobulin (Ig)-V like domain homologous to B7family ligands such as PD-L1, PD-L2, B7-H3 and B7-H4, as well as to theB7 family receptors (i.e., PD-1, CTLA-4, CD28, BTLA, ICOS) (FIG. 1B-C).Although the sequence identity of the Ig-V domains between B7 familyligands and receptors in general is not very high (<40%), the Ig-Vdomain of 4632428N05Rik bears the highest homology with B7 familyligands PD-L1 and PD-L2. Sequence alignment also reveals several highlyconserved cysteines (FIG. 1B) that are important for intra-chaindisulfide bond formation, which is characteristic of the B7 familyligands. See also FIG. 23; Sica, et al. (2003) Immunity 18: 849-861.

The extracellular domain of 4632428N05Rik contains only the Ig-V domainbut lacks the Ig-C domain (FIG. 1B-C). This unique feature ischaracteristic of the B7 family receptors, and distinguishes4632428N05Rik from all other B7 family ligands, which contain both Ig-Vand Ig-C domains. Freeman (2008) Proc Natl Acad Sci USA 105:10275-10276; Lazar-Molnar, et al. (2008) Proc Natl Acad Sci USA 105:10483-10488; Lin, et al. (2008) Proc Natl Acad Sci USA 105: 3011-3016;Schwartz, et al. (2001) Nature 410: 604-608; Stamper, et al. (2001)Nature 410: 608-61. Consistently, the phylogenic analysis using PhyMLalgorithm (Phylogenetic Maximum Likelihood) placed 4632428N05Rik in acloser evolutionary distance with B7 family receptors, in particularwith PD-1, than the B7 family ligands (FIG. 2). Guindon & Gascuel (2003)Syst Biol 52: 696-704. However, the cytoplasmic tail of VISTA (PD-L3)does not contain any signaling domains (e.g. ITIM, ITAM or ITSM), whichare the signature domains of B7 family receptors. Sharpe & Freeman(2002) Nat Rev Immunol. 2: 116-126. Despite its close evolutionaryrelationship with the inhibitory receptor PD-1, 4632428N05Rik representsa novel member of the B7 ligand family. Based on these structural andphylogenic characteristics, this molecule was named PD-1-eXpressed asLigand (VISTA (PD-L3)). VISTA (PD-L3) is also highly conserved betweenthe mouse and human orthologs, sharing 77% sequence identity (FIG. 1D).

The nucleic acid sequence encoding mouse VISTA (PD-L3) is set forthherein as SEQ ID NO:1 and the mouse VISTA (PD-L3) protein sequence isset forth as SEQ ID NO:2.

The human homolog of VISTA (PD-L3) is located on chromosome 10 (72.9 Mb)and composed of 6 exons thereby generating a transcript of 4689 bases inlength coding for a 311 residue protein. The human homolog mRNA codingsequence is provided in GENBANK accession number NM_022153 and proteinsequence give as NP_071436. The nucleic acid sequence encoding humanVISTA (PD-L3) is set forth herein as SEQ ID NO: 3 and the human VISTA(PD-L3) protein sequence is set forth as SEQ ID NO:4. Mouse and humangenes share 74% homology and are 68% identical at the protein level.Homologs were also identified in Rattus norvegicus on chromosome 20(27.7 Mb; GENBANK accession number BC098723), as well as Fugu rubripesand Danio rerio. In one embodiment, VISTA (PD-L3) proteins of thepresent share the common amino acid sequence set forth in SEQ ID NO: 5.Additional orthologues of VISTA have been identified and are shown inFIG. 23D, e.g., (SEQ ID NO: 17), human (SEQ ID NO: 16), kangaroo (SEQ IDNO: 18), dolphin (SEQ ID NO: 19), chicken (SEQ ID NO: 20), xenopus (SEQID NO: 21), zebra finch (SEQ ID NO: 22), zebrafish, and fugu (SEQ ID NO:23).

Example 2 Expression Studies of VISTA (PD-L3) by RT-PCR Analysis andFlow Cytometry

RT-PCR analysis was used to determine the mRNA expression pattern ofVISTA (PD-L3) in mouse tissues (FIG. 3A). VISTA (PD-L3) is mostlyexpressed on hematopoietic tissues (spleen, thymus, bone marrow), ortissues with ample infiltration of leukocytes (i.e. lung). Weakexpression was also detected in non-hematopoietic tissues (i.e. heart,kidney, brain, and ovary). Analysis of several hematopoietic cell typesreveals expression of VISTA (PD-L3) on peritoneal macrophages, splenicCD11b+ monocytes, CD11c+ DCs, CD4+ T cells and CD8+ T cells, but lowerexpression level on B cells (FIG. 3B). This expression pattern is alsolargely consistent with the GNF (Genomics Institute of Novartis ResearchFoundation) gene array database, as well as NCBI GEO (gene expressionomnibus) database (FIG. 4A-D). See Su, et al. (2002) Proc Natl Acad SciUSA 99: 4465-4470.

In order to study the protein expression, VISTA (PD-L3) specific hamster8D8 and 6E7 monoclonal antibodies were produced. The specificity isdemonstrated by positive staining on VISTA (PD-L3)-overexpressing murineEL4 T cells, but negative staining on PD-L1-overexpressing EL4 cells(FIG. 5).

Both polyclonal and monoclonal antibodies were raised against VISTA(PD-L3). Using a rabbit anti-VISTA (PD-L3) antibody, VISTA (PD-L3)protein was localized to lymphoid organs and prominently found in braintissue. Of the monoclonal antibodies identified, the specificity ofαVISTA (PD-L3) clone 8D8 was further evaluated. In this analysis, clone8D8 was tested for binding against a panel of PD-L like-Ig fusionprotein molecules including CTLA-4, PD-1, PD-L1, PD-L2, B7-1, B7-2,VISTA (PD-L3) and hlg. The results of this analysis indicated that 8D8aPDL-3 was highly specific for VISTA (PD-L3).

Specifically, using the anti-VISTA (PD-L3) monoclonal antibody clone8D8, VISTA (PD-L3) expression was analyzed on hematopoietic cells byflow cytometry. Foxp3GFP knock-in reporter mice were used to distinguishCD4+ nTregs. In peripheral lymphoid organs (spleen and lymph nodes),significant expression is seen on all CD4+ T cell subsets (see totalCD4+ T cells, or Foxp3-naïve T cells and Foxp3+ nTreg cells, and memoryCD4+ T cells), whereas CD8+ T cells express markedly lower amount ofsurface VISTA (PD-L3) (FIG. 3C). In thymus, VISTA (PD-L3) expression isnegative on CD4+CD8+ double positive thymocytes, low on CD4 singlepositive cells, and detectable on CD8 single positive cells. Next, astrong correlation of high VISTA (PD-L3) expression with CD11b markercan be seen for both splenic and peritoneal cells, including both F4/80macrophages and myeloid CD11c+ DCs (FIG. 3D-E). On the other hand, Bcells and NK cells are mostly negative for VISTA (PD-L3) expression. Asmall percentage of Gr-1+ granulocytes also express VISTA (PD-L3) (FIG.3F).

A differential expression pattern is shown on the same lineage of cellsfrom different lymphoid organs (FIG. 3G). For CD4+ T cells and CD11bintermediate monocytes, the expression level follows the pattern ofmesenteric lymph node>peripheral LN and spleen>peritoneal cavity andblood. This pattern is less pronounced for CD11bhi cells. This datasuggests that VISTA (PD-L3) expression on certain cell types might beregulated by cell maturity and/or tissue microenvironment.

In addition to freshly isolated cells, VISTA (PD-L3) expression wasanalyzed on splenic CD4+ T cells, CD11bhi monocytes and CD11c+ DCs uponin vitro culture with and without activation (FIG. 6). Spleen cells wereeither cultured with medium, or with anti-CD3 (for activating T cells),or with IFNγ and LPS (for activating monocytes and DCs) for 24 hrsbefore being analyzed for the expression of VISTA (PD-L3) and other B7family ligands (e.g. PD-L1, PD-L2, B7-H3 and B7-H4). This comparisonrevealed distinctive expression patterns between these molecules. VISTA(PD-L3) expression is quickly lost on all cell types upon in vitroculture, regardless of the activation status. In contrast, PD-L1expression is upregulated on CD4+ T cells upon stimulation, or onCD11bhi monocytes and CD11c+ DCs upon culture in medium alone, andfurther enhanced in the face of stimulation. The expression of PD-L2,B7-H3 and B7-H4 are not prominent under the culture conditions used. Theloss of VISTA (PD-L3) expression in vitro is unique when compared toother B7 family ligands, but might reflect non-optimal cultureconditions that fail to mimic the tissue microenvironment.

To address how VISTA (PD-L3) expression might be regulated in vivo, CD4TCR transgenic mice D011.10 were immunized with the cognate antigenchicken ovalbumin (OVA) emulsified in complete Freund's adjuvant (CFA).At 24 hrs after immunization, cells from the draining lymph node wereanalyzed for VISTA (PD-L3) expression (FIG. 7A). Immunization withantigen (CFA/OVA) but not the adjuvant alone drastically increased theCD11b+ VISTA (PD-L3)+ myeloid cell population, which contained a mixedpopulation of F4/80+ macrophages and CD11c+ DCs. Further comparison withPD-L1 and PD-L2 reveals that even though PD-L1 has the highestconstitutive expression level, VISTA (PD-L3) is the most highlyupregulated during such an inflammatory immune response (FIG. 7B).Collectively, these data strongly suggest that the expression of VISTA(PD-L3) on myeloid APCs is tightly regulated by the immune system, whichmight contribute to its role in controlling immune responses andregulating T cell immunity.

In contrast to its increased expression on APCs, VISTA (PD-L3)expression is diminished on activated DO11.10 CD4+ T cells at a latertime point upon immunization (i.e. at 48 hr but not at 24 hr) (FIG. 8).This result suggests that VISTA (PD-L3) expression on CD4 T cells invivo may be regulated by its activation status and cytokinemicroenvironment during an active immune response.

Example 3 Functional Impact of VISTA (PD-L3) Signaling on CD4+ and CD8+T Cell Responses

A VISTA (PD-L3)-Ig fusion proteins were was produced to examine theregulatory roles of VISTA (PD-L3) on CD4+ T cell responses. The VISTA(PD-L3)-Ig fusion protein contains the extracellular domain of VISTA(PD-L3) fused to the human IgG1 Fc region. When immobilized on themicroplate, VISTA (PD-L3)-Ig but not control Ig suppressed theproliferation of bulk purified CD4+ and CD8+ T cells in response toplate-bound anti-CD3 stimulation, as determined by arrested celldivision (FIG. 9A-B). The VISTA (PD-L3) Ig fusion protein did not affectthe absorption of anti-CD3 antibody to the plastic wells, as determinedby ELISA, thus excluding the possibility of non-specific inhibitoryeffects. PD-1 KO CD4+ T cells were also suppressed (FIG. 9C), indicatingthat PD-1 is not the receptor for VISTA (PD-L3). The inhibitory effectof PD-L1-Ig and VISTA (PD-L3)-Ig was also directly compared (FIG. 10).When titrated amounts of Ig fusion proteins were absorbed to themicroplates together with αCD3 to stimulate CD4+ T cells, VISTA(PD-L3)-Ig showed similar inhibitory efficacy as PD-L1-Ig fusionprotein.

Since bulk purified CD4+ T cells contain various subsets, the impact ofVISTA (PD-L3)-Ig on sorted naïve (CD25-CD44lowCD62Lhi) and memory(CD25-CD44hiCD62Llow) CD4+ T cell subsets was evaluated (FIG. 11). VISTA(PD-L3) can suppresses the proliferation of both subsets, albeit withmuch less efficacy on the memory cells.

To further understand the mechanism of VISTA (PD-L3)-mediatedsuppression, the expression of early TCR activation markers andapoptosis were measured following T cell activation in the presence orabsence of VISTA (PD-L3)-Ig. Consistent with the negative impact on cellproliferation, there is a global suppression on the expression of theearly activation markers CD69, CD44, and CD62L (supplemental FIG. 12A).On the other hand, the VISTA (PD-L3)-Ig fusion protein did not induceapoptosis. On the contrary, less apoptosis (as determined by thepercentage of annexin V+ 7AAD− cells) was seen in the presence of VISTA(PD-L3) or VISTA-Ig than the control-Ig, at both early (24 hr) and laterstage (48 hr) of TCR activation (FIG. 12B). For example, at 24 hr timepoint, on total “ungated’ population, ˜27% cells were apoptotic in thepresence of VISTA (PD-L3) or VISTA-Ig, but ˜39% control cells wereapoptotiC When examining the cells within the live cell R1 gate, it isapparent that VISTA (PD-L3) or VISTA-Ig strongly inhibitedactivation-induced-cell-death (ACID), because about 72.6% control cellsbecame apoptotic whereas only 43.5% cells were apoptotic when treatedwith VISTA (PD-L3) or VISTA-Ig. Similar results were seen for the 48 hrtime point. Therefore, it appears that VISTA (PD-L3) or VISTA negativelyregulates CD4+ T cell responses by suppressing early TCR activation andarresting cell division, but with minimum direct impact on apoptosis.This mechanism of suppression is similar to that of B7-H4. Sica, et al.(2003) Immunity 18: 849-861.

A 2-step assay was developed to determine whether VISTA (PD-L3) orVISTA-Ig can suppress pre-activated CD4 T cells, and how persistent itssuppressive effect is. It is shown that the suppressive effect of VISTA(PD-L3) or VISTA-Ig fusion protein persists after its removal at 24 hrpost activation (FIG. 9D). In addition, both naïve and pre-activatedCD4+ T cells could be suppressed by VISTA (PD-L3) or VISTA-Ig. See FIGS.9D(i), 9D(iii), and 9D(iv).

Next, the impact of VISTA (PD-L3) or VISTA-Ig on CD4+ T cell cytokineproduction was analyzed. VISTA (PD-L3) or VISTA-Ig suppressed theproduction of Th1 cytokines IL-2 and IFNα from bulk purified CD4+ T cellculture (FIG. 13A-B). The impact of VISTA (PD-L3) or VISTA was furthertested on separate naïve (CD25-CD44lowCD62Lhi) and memory(CD25-CD44hiCD62Llow) CD4+ T cell populations. It is shown that memoryCD4+ T cells are the major source for cytokine production within theCD4+ T cell compartment, and VISTA (PD-L3) or VISTA can suppress thisproduction (FIG. 13C-D). Similar inhibitory effect of VISTA (PD-L3) orVISTA on IFNα production from CD8+ T cells was also shown (FIG. 13E).This inhibitory effect of VISTA (PD-L3) or VISTA on cytokine productionby CD4+ and CD8+ T cells is consistent with the hypothesis that VISTA(PD-L3) or VISTA is an inhibitory ligand that down-regulates immuneresponses.

Next, studies were designed to determine the factors that are able toovercome the inhibitory effect of VISTA (PD-L3) or VISTA. Given thatVISTA (PD-L3) or VISTA suppressed IL-2 production, and IL-2 is criticalfor T cell survival and proliferation, IL-2 might circumvent theinhibitory activity of VISTA (PD-L3) or VISTA. As shown in FIG. 14A,exogenous IL-2, but not IL-15, IL-7, or IL-23, partially reversed thesuppressive effect of VISTA (PD-L3) or VISTA-Ig on cell proliferation.The incomplete rescue by high levels of IL-2 indicates that VISTA(PD-L3) or VISTA signaling targets broader T cell activation pathwaysthan simply IL-2 production. On the other hand, potent co-stimulationsignal provided by anti-CD28 agonistic antibody completely reversedVISTA (PD-L3) or VISTA-Ig mediated suppression (FIG. 14B), whereasintermediate levels of costimulation is still suppressed by VISTA(PD-L3) or VISTA signaling (FIG. 14C). This result suggests that VISTA(PD-L3) or VISTA-mediated immune suppression would be more effectiveunder less inflammatory conditions, but will be inevitably overwhelmedby strong positive costimulatory signals. In this regard, VISTA (PD-L3)or VISTA shares this feature with other suppressive B7 family ligandssuch as PD-L1 and B7-H4. Sica, et al. (2003) Immunity 18: 849-861;Carter, et al. (2002) Eur J Immunol. 32: 634-643.

In addition to VISTA (PD-L3) or VISTA-Ig fusion protein, it is necessaryto confirm that VISTA (PD-L3) or VISTA expressed on APCs can suppressantigen-specific T cell activation during cognate interactions betweenAPCs and T cells. For this purpose, VISTA (PD-L3) or VISTA-RFP or RFPcontrol protein was over-expressed via retroviral transduction in anartificial antigen presenting cell line (CHO-APC) that stably expressesMHCII and B7-2 molecules Latchman, et al. (2001) Nat Immunol 2: 261-268.One problem in expressing VISTA (PD-L3) or VISTA in CHO is that themajority of VISTA (PD-L3) or VISTA failed to localize to the cellsurface, perhaps due to the alien environment that lacks support forVISTA (PD-L3) or VISTA surface localization. Although there are no clearmotifs present on the cytoplasmic tail of VISTA (PD-L3) or VISTA tosuggest the mode of regulation, the tail might play a role for itsintracellular localization. Consequently, a tail-less VISTA (PD-L3) orVISTA mutant was designed and was found to successfully localize to CHOcell surface.

To stimulate T cell response, CHO-VISTA (PD-L3) or VISTA or CHO-RFPcells were incubated together with DO11.10 CD4+ T cells in the presenceof antigenic OVA peptide. As shown in FIG. 15A-C, CHO-VISTA (PD-L3) orVISTA induced less proliferation of D011.10 cells than CHO-RFP cells.This suppressive effect is more pronounced at lower peptideconcentrations, consistent with the notion that a stronger stimulatorysignal would overcome the suppressive impact of VISTA (PD-L3) or VISTA.

In addition, the inhibitory effect of full-length VISTA (PD-L3) or VISTAon natural APCs was confirmed. in vitro cultured bone marrow deriveddendritic cells (BMDC) do not express high level of VISTA (PD-L3) orVISTA (FIG. 16). VISTA (PD-L3) or VISTA-RFP or RFP was expressed inBMDCs by retroviral transduction during the 10 day culture period.Transduced cells were sorted to homogeneity based on RFP expression. Theexpression level of VISTA (PD-L3) or VISTA on transduced DCs wasestimated by staining with anti-VISTA (PD-L3) or VISTA monoclonalantibody, and found to be similar to the level on freshly isolatedperitoneal macrophages, thus within the physiological expression range(FIG. 16). Sorted BMDCs were then used to stimulate OVA-specifictransgenic CD4+ T cells (OTII) in the presence of OVA peptide (FIG.15D). Expression of VISTA (PD-L3) or VISTA on BMDCs suppressed thecognate CD4+ T cell proliferative responses. This result is consistentwith previous data using VISTA (PD-L3) or VISTA-Ig fusion protein andCHO-APC cells, suggesting that VISTA (PD-L3) or VISTA can suppress Tcell-mediated immune responses.

Example 4 Evaluation of Anti-VISTA (PD-L3) or VISTA Antibodies inMultiple Sclerosis Animal Model (EAE)

Because the αVISTA (PD-L3) or VISTA monoclonal antibodies in vivoappeared to suppress T cell responses, αVISTA (PD-L3) or VISTA wastested to evaluate if it can inhibit a T cell-mediated autoimmunedisease. Using the Experimental Allergic Encephalomyelitis (EAE) model,the functional impact of aPDL-L3 monoclonal antibodies on inflammatorydiseases was determined EAE is a widely used murine model of the humanautoimmune disease multiple sclerosis. EAE can be induced by eitherimmunization with myelin antigens in adjuvant or by adoptive transfer ofmyelin-specific T cells, which results in inflammatory infiltrates ofvarious effector T cells and B cells, and macrophages, and demyelinationof central nervous systems.

αPDL-L3 monoclonal antibody was tested in the passive EAE model to avoidinduction of anaphylaxis due to the injection of large amount ofmonoclonal antibody as foreign antigen. In this adoptive transfer EAEmodel, donor SJL mice were immunized with CFA and PLP peptide. On day10, total lymphocytes from draining LN were isolated, and cultured invitro with PLP peptide, IL-23 (20 ng/ml) and anti-IFNγ (10 μg/ml) for 4days. Expanded CD4 T cells were then purified and adoptively transferredinto naïve recipient mice. This analysis indicated that aPDL-L3monoclonal antibody delayed disease onset, as well as reduced diseaseseverity, thereby shifting the disease progression curve significantly(FIG. 17). In addition, it reduced severity in a large percentage of themice and greatly increased survival from around 22% to over 75%. Thisdemonstrated activity of aPDL-L3 monoclonal antibody in EAE isconsistent with the in vitro data, and demonstrates the use of thisreagent as a novel immunoregulatory reagent in various inflammatorydiseases.

Example 5 Expression of VISTA in the CNS

The expression of VISTA in the CNS was also effected. These assaysrevealed that in mice with disease, VISTA expression is markedly reduced(from 76%→33%) on the CD11b+ cells (FIG. 22), consistent with thehypothesis that the loss of VISTA may be permissive for enhancedinflammation. This is interesting, and likely functionally importantwhen we contrast inflammatory myeloid cells herein, with the MDSC intumors that express extremely high levels of VISTA. It has been reportedthat EAE mice have elevated numbers of myeloid derived suppressor cells(CD11b+Ly-6Chigh MDSC) in the spleen which are potently suppressive forT cell activation and may temper disease32. Our data strongly suggestthat VISTA may play a role in myeloid-mediated suppression in EAE.

Example 6 The Impact of VISTA on the Fate and Function of T Cells in EAE

We also conducted experiments assaying the effect of VISTA on the fateand function of T cells in EAE. We wanted to assess if VISTA alters thedevelopment of pathogenic, encephalitogenic T cells, clonal T cellexpansion, T cell polarity, longevity, and conversion of Teff→Treg. Westudied the impact of VISTA blockade on T cell fate in EAE. Consistentwith the higher disease score, analysis of CNS at the end of diseasecourse confirmed significantly more IL17A-producing CD4+ T cellinfiltration (from 0.66→11%) in 13F3 (αVISTA) treated group.

Example 7 VISTA (PD-L3) or VISTA Transgenic and Knock-Out Mice

Using Lentiviral infection of embryos, four transgenic mice ubiquitouslyexpressing VISTA (PD-L3) or VISTA have been produced. These mice expressfull-length VISTA (PD-L3) or VISTA under the control of the humanelongation factor 1 promoter. These mice were generated using lentiviralvector pWPT. Similar to other PD-L1 family members (Appay, et al. (2002)J. Immunol. 168: 5954-8), it is contemplated that VISTA (PD-L3) or VISTAwill function as a negative regulator in vivo while functioning toco-stimulate αCD3 T cell proliferation in vitro. In this respect, thesemice are expected to spontaneously develop autoimmunity and in vivoimmune responses in the VISTA (PD-L3) or VISTA transgenic mice (i.e.,humoral immune responses, T cell priming) are evaluated to assesssystemic autoimmune disease development.

For knock-out mice, VISTA (PD-L3) or VISTA is inactivated by homologousrecombination. A BAC clone containing full-length VISTA (PD-L3) or VISTAsequence was purchased from INVITROGEN® (Carlsbad, Calif.). A VISTA(PD-L3) or VISTA targeting vector was generated by inserting a 1.6 kbfragment located at the 5′ side of the second exon of VISTA (PD-L3) orVISTA gene upstream the neomycin gene and the 5 kb fragment located atthe 3′ side of the third exon of VISTA (PD-L3) or VISTA gene downstreamthe neomycin gene. B6-derived embryonic stem (ES) cells areelectroporated with VISTA (PD-L3) or VISTA targeting vector andrecombined clones are selected. Selected clones are then injected intoC57BL/6 blastocysts and the resulting chimeric male offspring are matedto FLP-deleter mice to remove the neomycin cassette. Transmission of thetargeted allele in the offspring is determined by PCR from genomic DNA.The second and the third exon contain the VISTA (PD-L3) or VISTA domain,therefore, the resulting mice have only the inactivated form of theVISTA (PD-L3) or VISTA molecule.

The overall immune capacity of VISTA (PD-L3) or VISTA deficient mice isdetermined as with other PD-L−/− mice, including assessment of T cellresponses to antigen, humoral immune responses, overt autoimmunity(e.g., Systemic Lupus Erythematosus, inflammatory bowel disease), andincreased susceptibility to induced autoimmune disease (experimentalautoimmune encephalomyelitis) (Chen (2004) supra).

Example 8 VISTA (PD-L3) or VISTA Specific Antibodies Tested inCollagen-Induced Arthritis Animal Model

Male DBA/1J mice were immunized at the base of their tail with 100 μl ofemulsion containing 100 μg chick type-II collagen (C-II) in CFA(mycobacterium tuberculosis 3.5 mg/ml) and boosted IP with 100 μgaqueous C-II on day 21 post-immunization. Mice of each treatment group(n=6) were either untreated (NT-black circles), injected with 300 μghamster IgG (Ham Ig-black squares) or injected with 300 μg ofmonoclonal-antibody “7c9” (red triangle) or “13F3” (green triangle), asindicated. Injections were given every 2 days. Arthritic swelling wasscored on a scale of 0-4 for each paw of each mouse on the daysindicated. The arthritis score shown is the total score of all paws ofmice in each treatment group divided by the number of mice in the group.

Example 9 VISTA Blockade by a Specific VISTA Monoclonal AntibodyEnhances T Cell Responses In Vitro

A VISTA-specific monoclonal antibody (13F3) was identified whichneutralizes VISTA-mediated suppression (FIG. 18). CD11b^(hi) myeloidAPCs were purified from naïve mice to stimulate OT-II transgenic CD4 Tcells in the presence or absence of 13F3. Consistent with itsneutralizing effect, 13F3 enhanced T cell proliferation stimulated byCD11b^(hi) myeloid cells, which were shown to express high levels ofVISTA.

Example 10 Anti-VISTA Enhances Anti-Tumor Immunity

Because of the capacity of anti-VISTA to enhance T cell activation,whether anti-VISTA would enhance the protective immune response to animmunogenic tumor was assessed. A model in which we have a great deal ofexperience is the bladder carcinoma, MB49. MB49 expresses male antigen,and thus it is modestly immunogenic in female mice, although, it willgrow and kill female mice if there is no immune intervention. To testthe efficacy of αVISTA therapy, female mice were administered MB49 tumorcells subcutaneously (sq) and treated with αVISTA. Days thereafter, thesize of the tumor was measured until the mice had to be euthanized. FIG.19 shows that anti-VISTA therapy greatly impairs tumor growth. This isdue to the ability of anti-VISTA to intensify cell-mediated immune (CMI)responses.

Example 11 Effect of AVISTA on Tumor Regression in 4 Murine Tumor Models

Experiments in the immunogenic bladder carcinoma tumor MB49 have shownthat neutralization of VISTA using monoclonal antibody 13F3 and protectshost from tumor growth. The data indicates that VISTA has a considerablenegative immunoregulatory role in the microenvironment of a tumorbecause of its extremely high expression of MDSCs. Studies examining theeffect of anti-mouse VISTA on the growth of immunogenic (MB49) andhighly non-immunogenic (B16) tumor models will further confirm theefficacy of αVISTA therapy, shed light on the mechanism of action, andprovide the basis for selecting the optimal dose and timing. Therationale for each tumor model is detailed below.

TABLE 3 Tumor Name Tumor Type Host Groups ASSAYS MB49 Bladder B6 FemaleαVISTA Tumor Carcinoma Control Ig growth MB49 Bladder B6 Male SurvivalCarcinoma Immune/ B16.F10 Melanoma B6 Male or female Autoimmune ID8Ovarian B6 Female Assays Cancer

MB49 in female mice: Efficacy in this murine model has beendemonstrated. MDSCs in this model also express elevated levels of VISTA.In this model, due to the presence of H—Y antigen, the MB49 tumor ismodestly immunogenic. Since we know anti-VISTA therapy is effective,this model will serve as a “positive” control to determine dosing (1-100μg/mouse; and timing (day of tumor inoculation, or 4, 7, 10 days aftertumor; therapeutic intervention) of anti-VISTA therapy.

MB49 in male mice: Using doses and timing effective in female mice, theefficacy of anti-VISTA therapy in male mice (in which the tumor is lessimmunogenic) is determined.

B16 melanoma: Anti-CTLA-4 monoclonal antibody was shown highly effectivein this model, and represents a non-immunogenic tumor where the mousemodel has been valuable for predicting success in humans. Dosing regimesand timing will be similar to those shown to be effective in the MB49model.

ID8 Ovarian carcinoma: It is in this model, that VISTA expression hasbeen shown to be extremely high on MDSCs. Mice bearing ID8 tumor aretreated with αVISTA at the time of tumor inoculum or at day 5, 15, 25post inoculation.

Methods. B6 WT mice are used to determine the optimal dose and timing ofanti-VISTA treatment for the remission of all murine tumor models noted.The models to be used are listed in the Table 3.

The readout for this dose and timing assay are tumor growth kinetics.For MB49 and B16 studies, all tumor studies are done via intradermal(i.d.) inoculation and therefore tumor size can be readily measured.Tumor measurements is collected every 2-3 days using a caliper. In eachof these models, the impact of anti-VISTA or control antibody will betested for its ability to slow tumor growth or facilitate tumorregression. Growth of ID8 will be followed using a luciferase transducedID8 and whole body imaging using an IVIS Workstation. In addition, hostsurvival will also be determined.

Data on tumor growth is expressed as mean tumor volume±SEM anddifferences between groups will be analyzed by two-tailed ANOVA.Probability (p) values less than 0.05 is considered statisticallysignificant. Survival data is analyzed using the Kaplan-Meier methodwith the Wilcoxon rank test and the log-rank test used to verify thesignificance of the difference in survival between groups. In the B16models, frequencies of mice that develop vitiligo is determined.

Using these methods slowed tumor growth and/or tumor regression in micetreated with anti-VISTA monoclonal antibody is obtained as compared withmice treated with control ab in several of the non-immunogenic tumormodels. It has already been shown that anti-VISTA treatment delays tumorgrowth in an immunogenic tumor model. As each of these tumor models havetheir own specific growth kinetics and, anticipated dependency on VISTAto confer tumor growth and suppress immunity, mice will be administeredmonoclonal antibody either at the time of tumor inoculum or at timesthereafter. Additionally, at least 3 different concentrations ofanti-VISTA monoclonal antibodies are tested to determine the optimaldose for therapeutic benefit.

As shown in FIG. 20A-E, VISTA monoclonal antibody treatment reducedtumor growth in all 4 of these tumor models wherein mice were inoculatedeither subcutaneously (sq) with (A) MB49, (B) MCA105, (C) EG7 tumorcells, or (D) intraperitoneal (ip) with ID8-luciferase tumor cells, andtreated with VISTA monoclonal antibody 13F3 every other day (300 μg)beginning on day +1. Subcutaneous tumor growth was monitored. ForID8-luciferase tumor, mice were imaged on day 30 using Xenogen IVIS. (E)VISTA expression on myeloid leukocytes in tumor-bearing mice was alsodetermined. Draining LN and tumor tissues (ascites) were analyzed forVISTA expression. These findings show that VISTA expressed on MDSC is amajor suppressive molecule that interferes with the development ofprotective anti-tumor immunity, and αVISTA relieves this suppressiveactivity allowing immune intervention and slowing growth of tumor. Thesefindings also support the conclusion that VISTA on myeloid cells inautoimmune disease plays a pivotal function in regulating the extent ofinflammation.

Example 12 Synthesis of Oligomeric VISTA and VISTA Fusion ProteinsUseful for the Treatment of Autoimmunity

Soluble VISTA-Ig in vitro is not suppressive nor can its binding tocells be readily detected. By contrast, this molecule bound to plasticis profoundly suppressive. In addition, studies using VISTA-Ig in vivodid not show overt activity. With respect to these studies the VISTA-Igthat was created has mutations in the CH2-CH3 domain precluding FcRbinding, and therefore is not cytophilic in vivo. Recent studies haveshown that tetrameric PD-L1 bound 100× higher (K_(d) 6×10⁻⁸ M) thanmonomeric PD-L126 to PD-1, and that binding to cells was readilydetectable. Tetrameric PD-L1 was not tested in vivo, but in vitro it wasshown to block the functional suppression by native PD-L1. Using similarmethods oligomers are made that will target the VISTA pathway and elicitpotent immunosuppressive activity in vitro ad in vivo.

Such oligomers are constructed using the monomeric extracellular domainof VISTA or a fragment thereof, e.g., at least 50, 75, 100, 125, 150,175 or 200 amino acids long which extracellular domain or a portionthereof is used as the building blocks for oligomer. In these methodsthe inventors take advantage of the well-established MHC tetramertechnologies. In these methods the VISTA ectodomain construct or afragment is linked to the N-terminus of a variety of oligomerizationdomains (identified herein) in order to generate a series of VISTAcomplexes with valencies that span from divalent to heptavalent.

Thereby, a series of non-covalent oligomers is created based on highaffinity coiled-coil domains that direct the stable formation ofdimeric, trimeric, tetrameric, pentameric and heptameric assemblies.These oligomeric constructs are expressed in a host cell (e.g., E.coli). When expression is effected in E. coli the expressed oligomersare then refolded and purified from inclusion bodies using standardlaboratory protocols. This approach has routinely produced high qualitymaterial for biological and structural analysis, including MHC-peptidecomplexes and trimeric GITRL66. The isolated oligomeric proteins arethen assessed by SDS-PAGE, analytical gel filtration, analyticalultracentrifugation and mass spectrometry. These quality controlmeasures ensure the availability of homogeneous, well-characterizedmaterials for in vitro and in vivo studies. The parallel organization ofthese constructs results in molecules in which the valency is equal tothe oligomeric state since each individual VISTA complex is positionedto productively interact with cell surface bound VISTA receptor. Theabove constructs possess extreme stability and homogeneity of oligomericstate. (Non-covalent coiled-coil oligomerization domains typicallyexhibit melting temperatures that exceed 100° C., except for theheptameter sequence which exhibits a melting temperature of 95° C.

In addition dimeric VISTA-Ig is tetramerized that is either cytophilicor not cytophilic. The Fc fusion constructs of VISTA in frame with theIgG1 Fc (both wild-type IgG1 and the existing non-FcR-binding IgG1) aremodified with an N-terminal BirA site for enzymatic biotinylation andcloned into the pIRES2-EGFP vector. Enzymatic biotinylation will allowspecific, single residue modification and orientation upon avidinmultimerization. This approach has been used for the generation ofnumerous Ig-fusion proteins, including B7-1, PD-L1, PD-L2 and TIM-3. Theexpressed proteins are then enzymatically biotinylated in vitro,purified by size exclusion HPLC, and tetramerized using PE-Avidin. Theresulting tetramers which are cytophilic or not, are assessed in vivo.

These engineered multimeric VISTA proteins are useful in treatingautoimmunity and other conditions wherein intervention in the VISTApathway and immunosuppression is therapeutically warranted.

Example 13 VISTA Adenoviral Vectors for Inducing Immune Suppression

Gene transfer using recombinant adeno-associated virus (AAV) has seengreat technological development in gene therapy Specifically,AAV-mediated gene delivery of PD-L1 gene, or CTLA4-Ig and CD40-Ig hasachieved therapeutic efficacy in autoimmune disease models of lupus(Kyttaris et al., 2005) and cardiac transplantation. These methods willbe used to deliver either full length VISTA, or oligomeric VISTAectodomains, and their therapeutic effects are assessed in the EAEmodel. Recombinant adenovirus vector expressing either full-lengthmurine VISTA, or oligomeric VISTA ectodomain, is created using theAdeno-XTM Expression System (Clontech) according to the manufacturer'sinstructions. Briefly, VISTA is cloned into an E1 and E3-deleted,pAdDEST-based expression vector, under the control of the humancytomegalovirus (CMV) promoter. VISTA and control lacZ expressingadenovirus are then purified from cell lysates. For systemicoverexpression of VISTA, adenovirusis administered to mice byintravenous tail vein injection (1×10⁹ plaque-forming units [Pfu])either prior to or shortly after disease induction via immunization, orafter disease onset. The control mice will receive 100 μl PBS. Diseasedevelopment and alterations are monitored in both SJL mice and C57BL/6mice, which exhibit different disease progression pattern, and whichrepresent two distinct forms of clinical manifestation of human MSpatients.

Example 14 Functional Studies with Engineered Proteins and AdenoviralVectors

Mice are also administered (5-100 μg of protein/mouse×3 weekly) withengineered VISTA and/or adenoviral vectors. Following administration, Tcell expansion, differentiation, as well as EAE development isdetermined.

Example 15 Structural Studies on VISTA and Determining MolecularDeterminants of VISTA Function

Affinity, specificity, oligomeric state, and the formation andlocalization of organized signaling complexes are critical contributorsto immune function. All of these features impact signaling and immuneregulation, as the organization of the receptor-ligand ectodomainsdirectly controls the recruitment, organization and function ofnon-covalently associated cytoplasmic signaling and scaffoldingmolecules. The high resolution crystal structure of VISTA is determinedusing techniques including bacterial, insect and mammalian expressionsystems, as well as high-throughput crystallization and structuredetermination approaches. To validate the crystallographically-observeddisulfide bonding pattern, high resolution mass spectrometry usingapproaches that successfully supported studies of TIM-3 and human DcR359will be used. Based on these structural results, a series of mutantswith altered oligomeric properties is designed, as well as mutants inthe vicinity of any perturbed regions of the VISTA IgV domain. Thesemutant proteins will provide additional direct mechanistic insight intoVISTA function and should be useful in therapeutics whereinimmunosuppression is desired such as the autoimmune, allergic andinflammatory diseases identified herein. These mutants, especiallyoligomers are tested in in vitro systems and are assessed in animalautoimmune and inflammatory disease models in order to assess theimmunosuppressive effect on disease progression, disease remission or inprotecting the animal from developing the autoimmune or inflammatorycondition.

These oligomeric VISTA proteins will activate the VISTA pathway andfunction as a target of immune intervention in autoimmunity. Thisintervention will suppress immunity and exert a therapeutic benefit onautoimmune disease and other conditions wherein autoimmune suppressionis desired. This is accomplished by administering the oligomerized VISTAproteins in different autoimmune and inflammatory models such as the EAEand collagen-induced arthritis animal models. In addition, as discussedabove, adenoviral vectors that over-express full-length VISTA or VISTAoligomers are constructed and tested in vivo. These studies will confirmthe immunosuppressive effects of VISTA oligomers.

Example 16 Experiments Using Conditional Over-Expressing VISTATransgenic Mouse Strain (VISTA Transgenic Mouse Strain: R26STOPFLVISTA(VISTA)

A targeting construct containing the full-length cDNA of VISTA precededby a loxP-flanked STOP cassette, has been targeted into the ubiquitouslyexpressed ROSA26 locus. Multiple correctly targeted R26StopFL/-VISTApups were born, and bred onto the CMV-Cre deleter strain60. Preliminarydata in the VISTA×CMV-cre confirm GFP and heightened VISTA expression.Studies on the immune status of these mice (T cell responses to antigen,antibody titers) will confirm a suppressed phenotype. The VISTA strainwill be interbred with CD4-cre, CD11c-cre, and Lys-Cre to determine ifthe lineage location of VISTA expression influences suppression. Thephenotype and function of the T cells is also determined and it isdetermined if over-expression of VISTA results in the generation ofaTreg. In these studies Tregs from OVA-immune cre×VISTA strain areadoptively transferred into WT hosts, to see if antigen immunization inthe presence of over-expressed VISTA induces antigen-specific Tregs.This should verify that VISTA impacts Treg differentiation.

In addition, studies are effected in the EAE model whereby the impact ofVISTA proteins□on different lineages (by interbreeding with CD4-,CD11c-, Lys-cre) with respect to disease development is assessed.Assuming that disease can be suppressed by lineage restrictedoverexpression of VISTA mutants or in the CMV×VISTA mutant□ the temporalcontrol of disease development is also using Cre-ERT2× VISTAη. Throughthe administration of tamoxifen we can induce overexpression of VISTAprior to, or at disease initiation or at peak disease to determine ifVISTA can impact on the induction and/or effector phases of immunity.Using BM chimeric mice, temporally-restricted overexpression of VISTAcan be restricted to the hematopoietic compartment. For an appreciationof controlling the window of time VISTA is overexpressed, VISTA isgenetically turned on, then serologically turned off with theadministration of anti-VISTA monoclonal antibody. These studies willdetermine where and when VISTA has to act to control the development andprogression of autoimmune disease.

Example 17 Effect of Anti-VISTA Antibodies CD40/TLR Agonist Vaccine

As shown in FIG. 21, experiments were conducted that assayed the effectof anti-VISTA antibodies on vaccine efficacy. These results show thatanti-VISTA enhances the therapeutic efficacy of a CD40/TLR vaccine.C57BL/6 mice were challenged with 1×10⁵ metastatic B16.F10 melanomacells s.q. Four days later, mice were vaccinated with 100 μg of thetumor associated antigen ΔV, 100 μg αCD40 FGK45 (CD40 agonisticantibody) and 100 μg S-27609(TLR7 agonist) with or without anti-VISTA(200 μg×3/week). Growth of tumor was monitored by caliper measurements.

Example 18 Expression Profiling

To facilitate comparisons with established expression profiles of Tregcells, standard growth and activation conditions were employed (McHugh,et al. (2002) supra). Briefly, fresh isolated Treg cells (˜96% positive)were inoculated at 106/mL into complete RPMI medium supplemented with10% fetal bovine serum and 100 units IL-2 in a 24-well plate precoatedwith anti-CD3 with or without anti-GITR (DTA-1) (Shimizu, et al. (2002)supra). The cells were cultured at 37° C. for 0 and 12 hours, RNA waspurified and subsequently analyzed using an AFFYMETRIX® mouse genomeA430 oligonucleotide array.

By comparing the data from resting or activated CD4+CD25+ T cell groups,gene expression patterns were found to be similar to those establishedin the art (Gavin, et al. (2002) supra; McHugh, et al. (2002) supra). Toidentify genes regulated by GITR signaling, gene expression profileswere compared between the different cell populations with or withoutanti-GITR treatment. A list of known as well as unknown genes werecompiled including the previously uncharacterized VISTA and Treg-sTNF.

Example 19 Molecular Cloning of VISTA, Retrovirus Production andRetroviral Transduction of Cells

Full length VISTA was cloned from purified murine CD4+ T cells. TotalRNA was isolated from CD4+ T cells using Qiagen RNAmini kit. cDNA wasgenerated using Bio-Rad iScript™ cDNA synthesis kit. Full-length VISTAwas amplified and cloned into the ECorI-XhoI site of a retroviral vectorpMSCV-IRES-GFP (Zhang & Ren (1998) Blood 92: 3829-3840) in which theIRES-GFP fragment was replaced by RFP, thus resulting in a fusionprotein of VISTA fused to the N-terminus of RFP. Helper freeretroviruses were generated in HEK293T cells by transient transfectionof the VISTA-RFP retroviral vector together with an ecotrophic packagingvector pCL-Eco (IMGENEX Corp.) Retroviral transduction of murine T cellline EL4 cells, or bone marrow derived DCs were carried out by spininfection at 2000 rpm at RT for 45 min in the presence of 8 μg/mlpolybrene (Sigma).

Example 20 Production of VISTA-Ig Fusion Protein

The extracellular domain of VISTA (amino acid 32-190) was amplified andcloned into the SpeI-BamHI sites of the parental vector CDM7B.Hollenbaugh, et al. (1995) J Immunol Methods 188: 1-7. This vectorcontains the mutant form of constant and hinge regions of human IgG1,which has much reduced binding to Fc receptors. The resulting vectorCDM7B-VISTA was co-transfected with a DHFR expression vector pSV-dhfr(McIvor & Simonsen (1990) Nucleic Acids Res 18: 7025-7032) into the CHO(dhfr-) cell line (ATCC #CRL-9096). Stable CHO cell clones that expressVISTA-Ig were selected in medium MEM-alpha without nucleotides(INVITROGEN®). Further amplification with 0.5-1 μM methotrexate (SIGMA®M9929) yielded clones expressing high levels of soluble VISTA-Ig fusionprotein. The fusion protein was further purified from culturesupernatant using standard protein-G column affinity chromatography.

Example 21 Generation of VISTA Monoclonal Antibodies

Armenian hamsters were immunized 4× times with EL4 cells over-expressingVISTA-RFP weekly, then boosted with VISTA-Ig fusion protein emulsifiedin CFA. Four weeks after the boost, hamsters were boosted again withsoluble VISTA-Ig fusion protein. Four days after the last boost, hamsterspleen cells were harvested and fused to the myeloma cell lineSP2/0-Ag14 (ATCC #CRL-1581) using standard hybridoma fusion techniquesShulman, et al. (1978) Nature 276: 269-270. Hybridoma clones that secretVISTA specific antibodies were selected after limiting dilution andscreened by both ELISA and flow cytometric methods.

Example 22 Inhibitory Activity of VISTA

The inhibitory activity of PD-L1 was revealed by using antigenpresenting cells over-expressing PD-L1 in vitro with CD4+ and CD8+ Tcell antigen receptor transgenic T cells and antigen stimulation(Carter, et al. (2002) Eur. J. Immunol. 32:634-43). Similarly, thelentivector disclosed herein, which expresses the full-length VISTA, istransduced into cell lines expressing class II major histocompatibilitycomplex (MHC) and class I MHC. The response of TEa Tg or the 2Ctransgenic T cells to antigen presented by empty vector-transduced orVISTA-transduced antigen presenting cells is determined according toestablished methods.

Example 23 Monoclonal Antibody Production

VISTA was overexpressed in the murine B cell line A20, and therecombinant cell line was used to immunize Armenian hamsters. After 5×cell immunization, hamsters were boosted with purified VISTA-Ig fusionprotein emulsified in CFA. Four weeks later, a final boost was providedwith soluble VISTA-Ig. Subsequently, fusions of hamster splenocytes withSP2/0 cells were performed on day 4. Sixteen different clones wereidentified that recognized VISTA-Ig fusion protein by ELISA, as well asstained VISTA but not PD-L1 overexpressed on the murine T cell line EL4.Eleven of the clones were successfully subcloned and prepared forevaluation of their ability to stain endogenous VISTA on cells andtissues, and to block VISTA functions.

Example 24 VISTA-Ig Conjugates Negatively Regulates T Cell Responses

The immunoglobulin (Ig) superfamily consists of many critical immuneregulators, including the B7 family ligands and receptors. VISTA, anovel and structurally distinct Ig superfamily inhibitory ligand, whoseextracellular domain bears homology to the B7 family ligand PD-L1. Thismolecule is designated V-domain Ig suppressor of T cell activation(VISTA). VISTA is primarily expressed on hematopoietic cells, and VISTAexpression is highly regulated on myeloid antigen-presenting cells(APCs) and T cells. A soluble VISTA-Ig fusion protein or VISTAexpression on APCs inhibits T cell proliferation and cytokine productionin vitro. A VISTA-specific monoclonal antibody interferes withVISTA-induced suppression of T cell responses by VISTA-expressing APCsin vitro. Furthermore, anti-VISTA treatment exacerbates the developmentof the T cell-mediated autoimmune disease experimental autoimmuneencephalomyelitis in mice. Finally, VISTA overexpression on tumor cellsinterferes with protective antitumor immunity in vivo in mice. Thesefindings show that VISTA, a novel immunoregulatory molecule, hasfunctional activities that are nonredundant with other Ig superfamilymembers and may play a role in the development of autoimmunity andimmune surveillance in cancer. See Wang, et al. (2011) The Journal ofExperimental Medicine 208(3): 577-592. In this Example, VISTA may alsobe referred to as “PD-XL.”

Materials and Methods

Mice.

C57BL/6 mice, OT-II CD4 transgenic mice, and SJL/J mice were purchasedfrom the Jackson Laboratory. FoxP3-GFP reporter mice were as previouslydescribed (Fontenot, et al. 2005) and were provided by A. Rudensky(University of Washington School of Medicine, Seattle, Wash.). PD-1 KOmice were provided by T. Honjo (Kyoto University, Kyoto, Japan;Nishimura, et al. 1999, 2001). All animals were maintained in apathogen-free facility at Dartmouth Medical School. All animal protocolswere approved by the Institutional Animal Care and Use Committee ofDartmouth College.

Antibodies, Cell Lines, and Reagents.

Antibodies α-CD3 (2C11), α-CD28 (PV-1), α-CD4 (GK1.5), α-CD8 (53-6.7),α-CD11b (M1/70), α-F4/80 (BM8), α-CD11c (N418), α-NK1.1 (PK136), α-Gr1(RB6-8C5), α-PD-L1 (MIN5), α-PD-L2 (TY25), α-B7-H3 (M3.2D7), and α-B7-H4(188) were purchased from eBioscience. LPS (Sigma-Aldrich), recombinantmouse IFN-γ (PeproTech), human IL-2 (PeproTech), and soluble PD-L1-Igfusion protein (R&D Systems) were used at the indicated concentrations.CFA and chicken OVA were purchased from Sigma-Aldrich. The B celllymphoma cell line A20 (BALB/c origin) was obtained from the AmericanType Culture Collection.

Molecular Cloning of VISTA, Retrovirus Production, and RetroviralTransduction of Cells.

Full-length VISTA was cloned from purified mouse CD4⁺ T cells. Total RNAwas isolated from CD4⁺ T cells using an RNAmini kit (QIAGEN). cDNA wasgenerated using an iScript cDNA synthesis kit (Bio-Rad Laboratories).Full-length VISTA was amplified and cloned into the ECORI-XhoI site of aretroviral vector pMSCV-IRES-GFP (Zhang and Ren, 1998), in which theIRES-GFP fragment was replaced by RFP, thus resulting in a fusionprotein of VISTA fused to the N terminus of RFP. Helper freeretroviruses were generated in HEK293T cells by transient transfectionof the VISTA-RFP retroviral vector together with an ecotrophic packagingvector pCL-Eco (Imgenex Corp.). Retroviral transduction of mouse T cellline EL4 cells or BMDCs was performed by spin infection at 2,000 rpm atroom temperature for 45 min in the presence of 8 μg/ml polybrene(Sigma-Aldrich).

Bioinformatics Analysis of VISTA.

Proteins that are evolutionarily related to the VISTA Ig-V sequence wereidentified by the BLAST algorithm (Altschul, et al. 1990). The mostsuitable structural templates from the Protein Data Bank (Berman, et al.2000) were identified with the mGenTHREADER algorithm (Lobley, et al.2009). PD-L1 (Protein Data Bank accession no. 3BIS), one of the topscoring hits, was selected as the template for comparative proteinstructure modeling. The structural model of VISTA was constructed withthe MMM server using the optimal combination of two alignment methods,MUSCLE and HHalign (Rai and Fiser, 2006; Rai, et al. 2006). 36 VISTAorthologous proteins were collected from the ENSEMBL database (Flicek,et al. 2008). Structure and sequence alignments were calculated withDALI (Holm and Park, 2000) and Clustalw (Larkin, et al. 2007),respectively, and were presented using the ESPript 2.2 server (Gouet, etal. 1999). The BLAST pairwise comparison network was constructed asdescribed previously (Atkinson, et al. 2009) and analyzed usingCytoscape (Shannon, et al. 2003).

Production of VISTA-Ig Fusion Protein.

The extracellular domain of VISTA (aa 32-190) was amplified and clonedinto the SpeI-BamHI sites of the parental vector CDM7B (Hollenbaugh, etal. 1995). This vector contains the mutant form of constant and hingeregions of human IgG1, which has much reduced binding to Fc receptors.The resulting vector CDM7B-VISTA was cotransfected with a dihydrofolatereductase expression vector pSV-dhfr (McIvor and Simonsen, 1990) intothe Chinese hamster ovary (dhfr⁻) cell line (#CRL-9096; American TypeCulture Collection). Stable Chinese hamster ovary cell clones thatexpress VISTA-Ig were selected in medium MEM-α without nucleotides(Invitrogen). Further amplification with 0.5-1 μM methotrexate (M9929;Sigma-Aldrich) yielded clones expressing high levels of soluble VISTA-Igfusion protein. The fusion protein was further purified from culturesupernatant using standard protein G column affinity chromatography.

Generation of VISTA Monoclonal Antibodies (mAb).

Armenian hamsters were immunized with EL4 cells overexpressing VISTA-RFPand then boosted with VISTA-Ig fusion protein emulsified in CFA. 4 wkafter the boost, hamsters were boosted again with soluble VISTA-Igfusion protein. 4 d after the last boost, hamster spleen cells wereharvested and fused to the myeloma cell line SP2/0-Ag14 (#CRL-1581;American Type Culture Collection) using standard hybridoma fusiontechniques (Shulman, et al. 1978). Hybridoma clones that secretVISTA-specific antibodies were selected after limiting dilution andscreened by both ELISA and flow cytometry methods.

RNA and RT-PCR.

Total RNA from various mouse tissue samples or purified hematopoieticcell types were collected by using TRIZOL® (Invitrogen) according to thecompany's instructions. cDNAs were prepared by using the iScript cDNAsynthesis kit (Bio-Rad Laboratories). Equal amounts of tissue cDNAs (10ng) were used for RT-PCR reactions to amplify full-length VISTA. PCRproducts were viewed after running through a 1% agarose gel.

Flow Cytometry and Analysis.

Flow cytometry analysis was performed on FACScan using CellQuestsoftware (BD). Data analysis was performed using FlowJo software (TreeStar, Inc.). To quantify cell proliferation, the histogram profile ofCFSE divisions was analyzed, and the percentage of proliferativeCFSE^(low) cells was graphed using Prism 4 (GraphPad Software, Inc.).

Cell Preparation.

Total CD4⁺ T cells were isolated from naive mice using a total CD4⁺ Tcell isolation kit (Miltenyi Biotec). When indicated, enriched CD4⁺ Tcells were flow sorted into naive (CD44^(low)CD25⁻CD62L^(hi)) and memory(CD44^(hi)CD25⁻CD62L^(low)) populations. For in vitro proliferationassays, CD4⁺ T cells were labeled with 5 μM CFSE (Invitrogen) for 10 minat 37° C. and washed twice before being stimulated.

For A20 assay, A20-RFP or A20-PD-XL cells (20,000) were pretreated with100 μg/ml mitomycin C (1 h) and then incubated with CFSE-labeled D011.10CD4⁺ T cells (100,000) in the presence of OVA peptide. Control-Ig or13F3 monoclonal antibody was added as indicated. Cell proliferation wasanalyzed at 72 h by CFSE dilution. For sorting CD11b^(hi) myeloid APCs,CD11b⁺ monocytes were enriched from naive splenocytes using CD11bmagnetic beads (Miltenyi Biotec). Total CD11b^(hi) myeloid APCs, orCD11b^(hi)CD11c⁻ monocytes and CD11b^(hi)CD11c⁺ myeloid DCs were sorted,irradiated (2,500 rad), and used to stimulate OT-II transgenic CD4⁺ Tcells in the presence of OVA peptide. Control-Ig or 13F3 monoclonalantibody was added as indicated. Cell proliferation was measured bytritium incorporation during the last 8 h of a 72-hour assay.

In Vitro Plate-Bound T Cell Activation Assay.

Purified CD4⁺ T cells (100,000 cells per well) were cultured in 96-wellflat-bottom plates in the presence of anti-CD3 (clone 2C11) and eitherVISTA-Ig or control-Ig at the indicated concentration ratios. Forexample, for a full-range titration, the 96-well plates were coated with2.5 μg/ml of α-CD3 mixed together with 1.25 μg/ml (ratio 2:1), 2.5 μg/ml(ratio 1:1), 5 μg/ml (ratio 1:2), or 10 μg/ml (ratio 1:4) VISTA-Ig orcontrol-Ig protein in PBS at 4° C. overnight. Wells were washed threetimes with PBS before adding CD4⁺ T cells. Replicate cultures were incomplete RPMI 1640 medium supplemented with 10% FBS, 10 mM Hepes, 50 μMβ-ME, and penicillin/streptomycin/L-glutamine. When indicated, either100 U/ml human IL-2 (PeproTech) or a titrated amount of α-CD28 (clonePV-1; Bio X Cell) was coated together with α-CD3 to rescue theinhibitory effects of VISTA-Ig. Cultures were analyzed on day 3 for CFSEprofiles or according to a time course as indicated.

Culture of BMDCs, Retroviral Transduction, and Stimulation of TransgenicCD4⁺ T Cells.

BMDCs were generated as described previously (Lutz, et al. 1999; Son, etal. 2002), with some modifications. In brief, on day 0, BM cells wereisolated from tibia and femur by flushing with a 27-gauge needle. Afterred blood cell lysis, 1-2×10⁶ BM cells were resuspended in 1 ml completeRPMI 1640 medium containing 20 ng/ml GM-CSF (PeproTech). Cells wereinfected with RFP or VISTA-RFP retrovirus in the presence of 8 μg/mlPolybrene (Sigma-Aldrich). Infection was performed by spinning the plateat 2,000 rpm for 45 min at room temperature. Cells were then culturedfor another 2 h before fresh medium was added. Similar infectionprocedure was repeated on days 1, 3, and 5. Loosely adherent cells (90%were CD11c⁺) were collected on day 10, and CD11c⁺RFP⁺-double positivecells were sorted and used to stimulate OT-II transgenic CD4⁺ T cells.For OT-II T cell proliferation assays, 100,000 CFSE-labeled OT-II CD4⁺ Tcells were cultured in 96-well round-bottom plates with 30,000 sortedRFP or VISTA-RFP BMDCs, with a titrated amount of synthetic OVA₃₂₃₋₃₃₉peptide (AnaSpec). Proliferation of OT-II T cells was analyzed at 72 hby examining CFSE profiles.

Tumor Experiment.

Parent MCA105 tumor cells were retrovirally transduced with VISTA-RFP orRFP control and sorted to homogeneity based on RFP expression. For tumorvaccination, naive C57BL/6 mice were immunized with 1,000,000 irradiatedMCA105 (10,000 rad) cells that were inoculated subcutaneously into theleft flank. On day 14, vaccinated mice were challenged with live MCA105tumor cells that were inoculated subcutaneously into the right flank.Tumor growth was monitored every 2 d. Mice were euthanized when tumorsize reached 150 mm². For T cell depletion, vaccinated mice werepretreated intraperitoneally (250 μg) with monoclonal antibody specificfor CD4⁺ T cells (clone GK1.5) and CD8⁺ T cells (clone 53.6.72) 2 dbefore live tumor cell challenge, and the treatment was repeated every3-4 d until the end of the experiment. Mice were euthanized when tumorsize reached 160 mm².

Passive Induction of EAE and Characterization of Central NervousSystem-Infiltrating CD4⁺ T Cells.

For passive transfer EAE, female SJL mice (6 wk old) were immunizedsubcutaneously with 200 μl of emulsion containing 400 μg Mycobacteriumtuberculosis H37Ra and 100 μg PLP peptide. Draining LN cells wereharvested on day 10 for in vitro stimulation. Red blood cells werelysed. Single cell suspensions (10,000,000 per microliter) were culturedin complete IMDM medium with 10% FBS, 50 μM 2-ME, 1 mM glutamine, 1%penicillin/streptavidin, 1 mM nonessential amino acids, 20 ng/ml IL-23,10 ng/ml IL-6, 10 ng/ml IL-10, 20 μg/ml anti-IFN-γ, and 20 μg/ml PLPpeptide. On day 4, cells were harvested, and live CD4 T cells werepurified using CD4 magnetic beads (Miltenyi Biotec). 1,500,000-2,000,000purified live CD4 T cells were adoptively transferred into naive SJLmice to induce EAE. Mice were treated with either nonspecific hamstercontrol-Ig or 400 μg VISTA-specific monoclonal antibody every 3 days.Disease was scored as the following: 0, no disease; 1, hind limbweakness or loss of tail tone; 2, flaccid tail and hind limb paresis;2.5, one hind limb paralysis; 3, both hind limb paralysis; 4, front limbweakness; 5, moribund. Mice were euthanized at a score of 4.

Cloning and Sequence and Structural Analysis of VISTA

Affymetrix® analysis of activated versus resting mouse CD25⁺ CD4⁺natural T_(reg) cells (nT_(reg) cells) revealed the expression of a geneproduct (RIKEN cDNA 4632428N05 or 4632428N05Rik) with unknown functionbut with sequence homology to the Ig superfamily. A 930-bp gene productwas cloned from the mouse CD4⁺ T cell cDNA library, which matched thepredicted size and sequence. Silico sequence and structural analysispredicts a type I transmembrane protein of 309 aa upon maturation. Itsextracellular domain contains a single extracellular Ig-V domain of 136aa, which is linked to a 23-aa stalk region, a 21-residue transmembranesegment, and a 97-aa cytoplasmic domain (FIG. 23A). The cytoplasmic tailof 4632428N05Rik does not contain any signaling domains. Based on thestructural feature of the Ig-V domain and its immune-suppressivefunction that is shown herein, this molecule was named VISTA.

A BLAST (Altschul, et al. 1990) sequence search with the VISTA Ig-Vdomain identified PD-L1 of the B7 family as the closest evolutionarilyrelated protein with a borderline significant e-value score of 10⁻⁴ andwith a sequence identity of 24%.

A structure-based sequence alignment of VISTA with the B7 family membersPD-L1, PD-L2, B7-H3, and B7-H4 highlights several amino acids that areknown to be systematically conserved in all Ig-V domain proteins and arethought to be important for the stability of the Ig-V fold (FIG. 23C).Examples include the two cysteines in the B and the F β strands thatform a disulfide bond between the two 13 sheets, which is a hallmarkfeature of Ig superfamily proteins (FIG. 23C). This multiple sequencealignment also reveals additional sequence features that are unique toVISTA.

Expression experiments of VISTA by RT-PCR analysis and flow cytometry.RT-PCR analysis was used to determine the messenger RNA expressionpattern of VISTA in mouse tissues (FIG. 3A). VISTA is mostly expressedon hematopoietic tissues (spleen, thymus, and BM) or tissues with ampleinfiltration of leukocytes (i.e., lung). Weak expression was alsodetected in nonhematopoietic tissues (i.e., heart, kidney, brain, andovary). Analysis of several hematopoietic cell types revealed expressionof VISTA on peritoneal macrophages, splenic CD11b⁺ monocytes, CD11c⁺DCs, CD4⁺ T cells, and CD8⁺ T cells but a lower expression level on Bcells (FIG. 3B). This expression pattern is also largely consistent withthe GNF (Genomics Institute of the Novartis Research Foundation) genearray database (symbol 4632428N05Rik; Su, et al. 2002), as well as theNational Center for Biotechnology Information GEO (Gene ExpressionOmnibus) database (Accession No. GDS868).

To study the protein expression, VISTA-specific hamster monoclonalantibodies were produced. The specificity is demonstrated by positivestaining on VISTA-overexpressing mouse EL4 T cells but negative stainingon PD-L1-overexpressing EL4 cells.

Using an α-VISTA monoclonal antibody clone 8D8, VISTA expression wasanalyzed on hematopoietic cells by flow cytometry. Foxp3-GFP knockinreporter mice were used to distinguish CD4⁺ nT_(reg) cells (Fontenot, etal. 2005). In peripheral lymphoid organs (spleen and LNs), significantexpression was seen on all CD4⁺ T cell subsets (see total CD4⁺ T cellsor Foxp3⁻ naive T cells and Foxp3 nT_(reg) cells and memory CD4⁺ Tcells), whereas CD8⁺ T cells expressed a markedly lower amount ofsurface VISTA (FIG. 3C). In thymus, VISTA expression was negative onCD4⁺CD8⁺-double positive thymocytes, low on CD4-single positive cells,and detectable on CD8-single positive cells. Next, a strong correlationof high VISTA expression with CD11b marker was seen for both splenic andperitoneal cells, including both F4/80 macrophages and myeloid CD11c⁺DCs (FIGS. 3D and 3E). In contrast, B cells and NK cells were mostlynegative for VISTA expression. A small percentage of Gr-1⁺ granulocytesalso expressed VISTA (FIG. 3F).

A differential expression pattern was shown on the same lineage of cellsfrom different lymphoid organs (FIG. 3G). For CD4⁺ T cells andCD11b^(intermediate) monocytes, the expression level followed thepattern of mesenteric LN>peripheral LN and spleen>peritoneal cavity andblood. This pattern was less pronounced for CD11b^(hi) cells. These datasuggest that VISTA expression on certain cell types might be regulatedby cell maturity and/or tissue microenvironment.

In addition to freshly isolated cells, VISTA expression was analyzed onsplenic CD4⁺ T cells, CD11b^(hi) monocytes, and CD11c⁺ DCs upon in vitroculture with and without activation (FIG. 6). Spleen cells were culturedwith medium, with α-CD3 (for activating T cells), or with IFN-γ and LPS(for activating monocytes and DCs) for 24 h before expression analysisof VISTA and other B7 family ligands (e.g., PD-L1, PD-L2, B7-H3, andB7-H4). This comparison revealed distinctive expression patterns betweenthese molecules. VISTA expression was quickly lost on all cell typesupon in vitro culture, regardless of the activation status. In contrast,PD-L1 expression was up-regulated on activated CD4⁺ T cells or onCD11b^(hi) monocytes and CD11c⁺ DCs after culture in medium alone andfurther enhanced upon stimulation. The expression of PD-L2, B7-H3, andB7-H4 was not prominent under the culture conditions used. The loss ofVISTA expression in vitro is unique when compared with other B7 familyligands but might reflect nonoptimal culture conditions that fail tomimic the tissue microenvironment.

To address how VISTA expression might be regulated in vivo, CD4 TCRtransgenic mice DO11.10 were immunized with the cognate antigen chickenOVA emulsified in CFA. At 24 h after immunization, cells from thedraining LN were analyzed for VISTA expression (FIG. 7A). Immunizationwith antigen (CFA/OVA) but not the adjuvant alone drastically increasedthe CD11b⁺ VISTA⁺ myeloid cell population, which contained a mixedpopulation of F4/80⁺ macrophages and CD11c⁺ DCs. Further comparison withPD-L1 and PD-L2 revealed that even though PD-L1 had the highestconstitutive expression level, VISTA was the most highly up-regulatedduring such an inflammatory immune response (FIG. 7B). Collectively,these data strongly suggest that the expression of VISTA on myeloid APCsis tightly regulated by the immune system, which might contribute to itsrole in controlling immune responses. In contrast to its increasedexpression on APCs, VISTA expression was diminished on activated DO11.10CD4⁺ T cells at a later time point upon immunization (i.e., at 48 h butnot at 24 h).

Functional impact of VISTA signaling on CD4⁺ and CD8⁺ T cell responsesin vitro. A VISTA Ig fusion protein (VISTA-Ig) was produced to examinethe regulatory roles of VISTA on CD4⁺ T cell responses. VISTA-Igcontained the extracellular domain of VISTA fused to the human IgG1 Fcregion. When immobilized on the microplate, VISTA-Ig but not control-Igsuppressed the proliferation of bulk purified CD4⁺ and CD8⁺ T cells inresponse to α-CD3 stimulation (FIGS. 9A and 9B). The VISTA-Ig did notaffect the absorption of anti-CD3 antibody to the plastic wells, asdetermined by ELISA, thus excluding the possibility of nonspecificinhibitory effects. The inhibitory effect of PD-L1-Ig and VISTA-Ig wasdirectly compared. When titrated amounts of Ig fusion proteins wereabsorbed to the microplates together with α-CD3 to stimulate CD4⁺ Tcells, VISTA-Ig showed potent inhibitory efficacy similar to thePD-L1-Ig fusion protein. PD-1 KO CD4⁺ T cells were also suppressed (FIG.9C), indicating that PD-1 is not the receptor for VISTA.

Because bulk purified CD4⁺ T cells contain various subsets, the impactof VISTA-Ig on sorted naive (CD25⁻ CD44^(low)CD62L^(hi)) and memory(CD25⁻CD44^(hi)CD62L^(low)) CD4⁺ T cell subsets was evaluated. VISTAsuppressed the proliferation of both subsets, albeit with less efficacyon the memory cells.

To further understand the mechanism of VISTA-mediated suppression, theexpression of early TCR activation markers and apoptosis were measuredafter T cell activation. Consistent with the negative effect on cellproliferation, there was a global suppression on the expression of theearly activation markers CD69, CD44, and CD62L (FIG. 12A). In contrast,VISTA-Ig did not induce apoptosis. Less apoptosis (as determined by thepercentage of annexin V⁺ 7AAD⁻ cells) was seen in the presence ofVISTA-Ig than the control-Ig at both early (24 hours) and later (48hours) stages of TCR activation (FIG. 12B). For example, at 24 h, of thetotal ungated population, ˜27% of cells were apoptotic in the presenceof VISTA-Ig, but ˜39% of cells were apoptotic in the presence ofcontrol-Ig. Similarly, of the cells within the live cell R1 gate, ˜72.6%cells became apoptotic in the presence of control-Ig, whereas only˜43.5% cells were apoptotic in the presence of VISTA-Ig. Similar resultswere seen at the 48-h time point. Therefore, it appears that VISTAnegatively regulates CD4⁺ T cell responses by suppressing early TCRactivation and arresting cell division but with minimum direct impact onapoptosis. This mechanism of suppression is similar to that of B7-H4(Sica, et al. 2003).

A two-step assay was developed to determine whether VISTA-Ig cansuppress preactivated CD4⁺ T cells and how persistent its suppressiveeffect is. The suppressive effect of VISTA-Ig fusion protein persistedafter its removal at 24 hours after activation (FIG. 9D, ii). Inaddition, both naive and preactivated CD4⁺ T cells were suppressed byVISTA-Ig (FIG. 9D, i, iii, and iv).

Next, the effect of VISTA-Ig on CD4⁺ T cell cytokine production wasanalyzed. VISTA-Ig suppressed the production of Th1 cytokines IL-2 andIFN-γ from bulk purified CD4⁺ T cell culture (FIGS. 13A and 13B). Theimpact of VISTA was further tested on separate naive(CD25⁻CD44^(low)CD62Lhi) and memory (CD25⁻CD44^(hi)CD62L^(low)) CD4⁺ Tcell populations. Memory CD4⁺ T cells were the major source for cytokineproduction within the CD4⁺ T cell compartment, and VISTA suppressed thisproduction (FIGS. 13C and 13D). IFN-γ production from CD8⁺ T cells wasalso inhibited by VISTA-Ig (FIG. 13E). This inhibitory effect of VISTAon cytokine production by CD4⁺ and CD8⁺ T cells is consistent with thehypothesis that VISTA is an inhibitory ligand that down-regulates Tcell-mediated immune responses.

Further experiments were designed to determine the factors that are ableto overcome the inhibitory effect of VISTA. Given that VISTA suppressedIL-2 production and IL-2 is critical for T cell survival andproliferation, we hypothesized that IL-2 might circumvent the inhibitoryactivity of VISTA. As shown in FIG. 14A, exogenous IL-2 but not IL-15,IL-7, or IL-23 partially reversed the suppressive effect of VISTA-Ig oncell proliferation. The incomplete rescue by high levels of IL-2indicates that VISTA signaling targets broader T cell activationpathways than simply IL-2 production. In contrast, potent co-stimulatorysignals provided by α-CD28 agonistic antibody completely reversedVISTA-Ig-mediated suppression (FIG. 14B), whereas intermediate levels ofco-stimulation continued to be suppressed by VISTA signaling (FIG. 14C).In this regard, VISTA shares this feature with other suppressive B7family ligands such as PD-L1 and B7-H4 (Carter, et al. 2002; Sica, etal. 2003).

In addition to the VISTA-Ig fusion protein, it was necessary to confirmthat VISTA expressed on APCs can suppress antigen-specific T cellactivation during cognate interactions between APCs and T cells. We haveused two independent cell systems to address this question. First,VISTA-RFP or RFP control protein was overexpressed via retroviraltransduction in a B cell line A20. The correct cells surfacelocalization of VISTA-RFP fusion protein was confirmed by fluorescencemicroscopy. To stimulate T cell response, A20-VISTA or A20-RFP cellswere incubated together with DO011.10 CD4⁺ T cells in the presence ofantigenic OVA peptide. As shown in FIGS. 15A and C), A20-VISTA inducedless proliferation of DO11.10 cells than A20-RFP cells. This suppressiveeffect is more pronounced at lower peptide concentrations, which isconsistent with the notion that a stronger stimulatory signal wouldovercome the suppressive impact of VISTA.

Second, the inhibitory effect of full-length VISTA on natural APCs wasconfirmed. in vitro cultured BM-derived DCs (BMDCs) did not express highlevels of VISTA (FIG. 16). VISTA-RFP or RFP was expressed in BMDCs byretroviral transduction during the 10-day culture period. Transducedcells were sorted to homogeneity based on RFP expression. The expressionlevel of VISTA on transduced DCs was estimated by staining with α-VISTAmonoclonal antibody and found to be similar to the level on freshlyisolated peritoneal macrophages, thus within the physiologicalexpression range (FIG. 16). Sorted BMDCs were then used to stimulateOVA-specific transgenic CD4⁺ T cells (OT-II) in the presence of OVApeptide. The expression of VISTA on BMDCs suppressed the cognate CD4⁺ Tcell proliferation (FIG. 15D). This result is consistent with data (FIG.5) using VISTA-Ig fusion protein or VISTA-expressing A20 cells,suggesting that VISTA expressed on APCs can suppress T cell-mediatedimmune responses.

To validate the impact of VISTA expression in vivo, whether VISTAoverexpression on tumor cells could impair the antitumor immune responsewas examined. MCA105 (methylcholanthrene 105) fibrosarcoma does notexpress VISTA. Two MCA105 tumor lines were established by retroviraltransduction with either VISTA-RFP or RFP control virus. Because MCA105tumor is immunogenic and can be readily controlled in hosts preimmunizedwith irradiated MCA105 cells (Mackey, et al. 1997), we examined theeffect of tumor VISTA expression on such protective immunity. As shownin FIG. 26A, VISTA-expressing MCA105 grew vigorously in vaccinatedhosts, whereas the control tumors failed to thrive. To confirm thatthere is no intrinsic difference in tumor growth rate in the absence ofT cell-mediated antitumor immunity, tumors were inoculated in vaccinatedanimals in which both CD4⁺ and CD8⁺ T cells were depleted usingmonoclonal antibodies. As shown in FIG. 26B, upon T cell depletion, bothMCA105RFP and MCA105VISTA tumors grew at an equivalent rate and muchmore rapidly than non-T-depleted hosts. Together, these data indicatethat VISTA expression on tumor cells can interfere with the protectiveantitumor immunity in the host. VISTA blockade by a specific monoclonalantibody enhanced T cell responses in vitro and in vivo.

A VISTA-specific monoclonal antibody (13F3) was identified to neutralizeVISTA-mediated suppression in the A20-DO11.10 assay system (FIG. 25A).To further confirm the impact of 13F3 on T cell responses, CD11b^(hi)myeloid APCs were purified from naive mice to stimulate OT-II transgenicCD4⁺ T cells in the presence or absence of 13F3 (FIG. 25B). Consistentwith its neutralizing effect, 13F3 enhanced T cell proliferationstimulated by CD11b^(hi) myeloid cells, which were shown to express highlevels of VISTA (FIG. 3). A similar effect of 13F3 could be seen on bothCD11b^(hi)CD11c⁺ myeloid DCs and CD11b^(hi)CD11c⁻ monocytes (FIG.25C-D).

Next, the impact of VISTA blockade by monoclonal antibody was examinedin a passive transfer model of EAE, which is a mouse autoimmuneinflammatory disease model for human multiple sclerosis (Stromnes andGoverman, 2006). Encephalitogenic CD4⁺ T cells were primed in the donormice by active immunization with proteolipid protein (PLP) peptide andadoptively transferred into naive mice. So as to carefully evaluate theability of α-VISTA to exacerbate disease, tittered numbers of activatedencephalitogenic T cells were passively transferred into naive hoststreated with α-VISTA or control-Ig, and the development of EAE wasmonitored. 13F3 was found to significantly accelerate disease onset, aswell as exacerbate disease severity under the suboptimal T cell transferdosage. The 13F3-treated group reached 100% disease incidence by day 14,whereas those mice treated with control antibody did not reach 100%disease incidence during the experimental duration. The mean diseasescore was significantly higher in the 13F3-treated group than thecontrol group throughout the disease course. Consistent with the higherdisease score, analysis of the central nervous system at the end ofdisease course confirmed significantly more IL-17A-producing CD4⁺ T cellinfiltration in the 13F3-treated group.

Example 25 VISTA KO Mice

Experiments were conducted where the expression of VISTA was knocked outin mice in order to further assess the effects of VISTA in vivo. It isknown to assess the effects of immunoregulatory molecules in vivo byknocking out the expression of such molecule and assessing its effect inthe knock-out animal versus wild-type mice. For example, CTLA4 KO miceshow dramatic loss of peripheral tolerance, massive lympho-proliferativephenotype, which results in early death, with this being explained bydifferent mechanisms of action including the direct suppression ofeffector T cell activation, and CTLA4⁺ involvement in the suppressivefunction of Foxp3+CD4+ Tregs and de novo induction of adaptive Tregs.

In addition, PD-1 KO mice have been produced in order to assess the invivo effects of PD-1. It has been observed that PD1 KO mice developmilder autoimmune phenotypes, which vary depending upon geneticbackground and are generally late-onset.

TABLE 1 Autoimmune phenotypes of Pdcd1^(−/−) mice Age at GenotypePhenotype onset Penetrance Refs C57BL/6-Pdcd1^(−/−) SLE-like >6 months~50% [29] BALB/c-Pdcd1^(−/−) DCM 5-25 weeks 10-60%^(a)  [30, 49]Gastritis 10-20 weeks ~80% [49] NOD-Pdcd1^(−/−) Diabetes 4-10 weeks 100%[33] BALB/c-Fcgr2b^(−/−) Hydrone- 10-20 weeks  35% [49] Pdcd1^(−/−)phrosis 2C-Pdcd1^(−/−) H-2^(b/d) GVH-like 5-10 weeks 26-100%^(b )  [29]^(a)The penetrance of dilated cardiomyopathy (DCM) is variable among thedifferent colonies of mice examined ([49] and our unpublishedobservations). ^(b)The penetrance of GVH-like disease is variabledepending on the genetic background (our unpublished observations).

In addition, PD-L1 KO mice have been made. These mice exhibit noapparent pathological phenotypes in aged mice; exhibit enhanced adaptiveimmune response upon immune challenge (i.e. model antigen immunization,infection etc); and the knockout of this protein results in thepromotion of autoimmune disease development in disease-prone background

Accordingly, a critical function of PD-L1 is in mediating peripheraltolerance at tissue sites due to the tissue expression of PD-L1 onnon-hematopoietic cell types.

As noted previously, VISTA bears homology to PD-L1. Similarly, withinthe hematopoietic compartment, VISTA is highly expressed on CD11bhighmyeloid cells under normal conditions and myeloid-derived suppressorcells (MDSC) in tumor-bearing hosts. VISTA is also expressed on CD4+ andCD8+ T cells, and Tregs. Moreover, as the human homologue shares 90%homology with murine VISTA and similar expression patterns, the in vivoeffects of human and murine VISTA should be functionally equivalent.

As shown in FIG. 42, VISTA-KO mice develop inflammatory phenotypes,including the enhanced amounts of serum inflammatory cytokines includingIP-10, MCP-1, MIG, IL-5 and eotoxin. Also, as shown in FIG. 43 thesesame mice exhibit an increase in spontaneously activated CD4+ and CD8+ Tcells within their serum. Also, as shown in FIG. 44, the blood T cellsof these VISTA-KO mice express increased amounts of inflammatorycytokines, i.e., the CD4+ T cells express increased amounts of gammainterferon and IL17A and the CD8+ T cells express increased amounts ofgamma interferon and TNFalpha. Further, as shown in FIG. 45, the spleenT cells similarly express inflammatory cytokines, i.e., the spleen CD4+T cells express increased amounts of gamma interferon, TNF-alpha andIL17A and the spleen CD8+ T cells express increased amounts of gammainterferon and TNFalpha.

After 1 year, the VISTA KO mice also exhibit evidence of organ specificimmune cell infiltration in different organs including the pancreas,lung, stomach. However, the KO mice do not otherwise exhibit any overtorgan-specific autoimmune pathology.

Accordingly, the results herein show that soluble VISTA-Ig fusionprotein, or VISTA expressed on APCs, acts as a ligand to suppress CD4+and CD8+ T cell proliferation and cytokine production, via a cognatereceptor independent of PD-1. Moreover, the results herein indicate thatVISTA functions as a novel immune checkpoint protein ligand: bycontrolling inflammation and autoimmunity and by impairing thegeneration of anti-tumor immunity.

Related thereto, the results herein indicate that VISTA-Ig fusionprotein may be used as a negative regulator of inflammation because itmay significantly reduce production of IL-10, TNFalpha and IFNgamma byCD4+ and CD8+ T cells. This should result in a therapeuticdownregulation of the immune response and provide relief from autoimmuneor inflammatory disorders.

Discussion

VISTA is as a novel member of the Ig superfamily network, which exertsimmunosuppressive activities on T cells both in vitro and in vivo and isan important mediator in controlling the development of autoimmunity andthe immune responses to cancer. The data presented suggests that (a)VISTA is a new member of the Ig superfamily that contains an Ig-V domainwith distant sequence similarity to PD-L1, (b) when produced as an Igfusion protein or overexpressed on artificial APCs, it inhibits bothCD4⁺ and CD8⁺ T cell proliferation and cytokine production, (c) VISTAexpression on myeloid APCs is inhibitory for T cell responses in vitro,(d) overexpression on tumor cells impairs protective antitumor immunityin vaccinated mice, and (e) antibody-mediated VISTA blockade exacerbatesthe development of a T cell-mediated autoimmune disease, EAE.

Bioinformatics analysis of the VISTA Ig-V domain suggests that theB7-butyrophilin family members PD-L1, PD-L2, and MOG, as well as thenon-B7 family CAR and VCBP3 are the closest evolutionary relatives ofVISTA (FIG. 23). However, close examination of primary sequencesignatures suggests that all VISTA orthologues share unique andconserved sequence motifs and that VISTA possibly represents astructurally and functionally novel member of the Ig superfamily.Specifically, the presence of four invariant cysteines that are uniqueto the VISTA ectodomain (three in the Ig-V domain and one in the stalk)may contribute to novel structural features that impact its function.Given their strict invariance, it is plausible that all fourVISTA-specific cysteines participate in disulfide bonds. Thisobservation suggests several possibilities, including that the fourcysteines (a) form two intramolecular disulfide bonds, (b) form fourintermolecular disulfide bonds at a dimer interface, and (c) form oneintramolecular and two intermolecular disulfide bonds. Any of thesescenarios would represent a novel disulfide bonding pattern and wouldlead to unique tertiary and/or quaternary structures relative to typicalIg superfamily members. In addition, a global sequence comparisonsuggests that VISTA is not a member of any known functional groupswithin the Ig superfamily.

The expression pattern of VISTA further distinguishes VISTA from otherB7 family ligands. This data has contrasted mostly with PD-L1 and PD-L2because of the higher sequence homology between these two ligands andVISTA and their similar inhibitory function on T cell activation. Thesteady-state expression of VISTA is selectively expressed onhematopoietic cells and most highly expressed on both APCs (macrophagesand myeloid DCs) and CD4⁺ T lymphocytes. In this context, PD-L1 hasbroad expression on both hematopoietic and nonhematopoietic cells,whereas PD-L2 is restricted on DCs and macrophages (Keir, et al. 2006,2008). Although both PD-L1 and PD-L2 are up-regulated on APCs upon invitro culture and upon activation (Yamazaki, et al. 2002; Liang, et al.2003; Keir, et al. 2008), VISTA expression on myeloid cells and T cellsis lost after short-term in vitro culture, regardless of whether anystimuli were present (FIG. 6). Such loss might reflect the necessaryrole of lymphoid tissue microenvironment to maintain or regulate VISTAexpression in vivo. Consistent with this hypothesis, even atsteady-state, VISTA is differentially expressed at different tissuesites (i.e., higher at mesenteric LN than peripheral lymphoid tissuesand lowest in blood). We speculate that such different expression levelsmight reflect the differential suppressive function of VISTA atparticular tissue sites.

VISTA expression in vivo is highly regulated during active immuneresponse. Immunization with adjuvant plus antigen (OVA/CFA) but notadjuvant alone (CFA) in TCR transgenic mice induced a population ofVISTA^(hi) myeloid APCs within the draining LN (FIG. 7). The need forantigen suggests that VISTA up-regulation on APCs might be a result of Tcell activation. Compared with VISTA, PD-L1 and PD-L2 were alsoup-regulated on myeloid APCs in response to immunization but to a muchlesser degree. We speculate that the induction of VISTA⁺ myeloid APCsconstitutes a self-regulatory mechanism to curtail an ongoing immuneresponse. Consistent with this hypothesis, a neutralizingVISTAmonoclonal antibody enhanced T cell proliferative response in vitrowhen stimulated by VISTA-expressing myeloid APCs (FIG. 25).

In contrast to the expression pattern on myeloid cells, VISTA expressionis diminished on in vivo activated CD4⁺ T cells. This result suggeststhat VISTA expression on CD4 T cells in vivo may be regulated by itsactivation status and cytokine microenvironment during an active immuneresponse. Such down-regulation is unique and has not been seen for otherinhibitory B7 family ligands such as PD-L1, PD-L2, and B7-H4. Althoughthe functional significance of VISTA expression on CD4⁺ T cells iscurrently unknown, the possibility of reverse signaling from T cells toAPCs during their cognate interaction will be investigated in futurestudies.

The inhibitory ligand function of VISTA was delineated by using theVISTA-Ig fusion protein, APCs expressing VISTA, and tumorsoverexpressing VISTA, as well as the neutralizingmonoclonal antibodyboth in vitro and in vivo. VISTA-overexpressing tumor could overcome apotent protective immunity in vaccinated hosts. The strong enhancingeffect of VISTAmonoclonal antibody in the EAE model further validatesthe hypothesis that VISTA is an inhibitory ligand in vivo. Similarapproaches have been used to characterize the functions of other B7family ligands (Sica, et al. 2003; Keir, et al. 2008). It is importantto note that VISTA exerts its suppressive function by engaging adifferent receptor than PD-1 (FIG. 9). The fact that blockade of theVISTA pathway exacerbates EAE confirms that its function is notredundant with PD-L1 or PD-L2. On the contrary, we speculate that VISTAcontrols immune response in a manner that is reflected by its uniquestructural features, expression pattern, and dynamics. Identification ofits unknown receptor will further shed light on the mechanisms ofVISTA-mediated suppression.

In summary, VISTA was identified as a novel immune-suppressive ligand.Expression of VISTA on APCs suppresses T cell responses by engaging itsyet to be identified counter-receptor on T cells during cognateinteractions between T cells and APCs. VISTA blockade enhanced Tcell-mediated immunity in an autoimmune disease model, suggesting itsunique and nonredundant role in controlling autoimmunity when comparedwith other inhibitory B7 family ligands such as PD-L1 and PD-L2. Itshighly regulated expression pattern at early stages of immune activationmight also indicate a feedback control pathway to down-regulate T cellimmunity and attenuate inflammatory responses. In this regard,therapeutic intervention of the VISTA inhibitory pathway represents anovel approach to modulate T cell-mediated immunity for treatingdiseases such as viral infection and cancer.

Example 25 The VISTA Pathway as a Target of Immune Intervention inAutoimmunity

The purpose of these studies is to determine if soluble VISTA-Igproteins can suppress immune responses in vivo. Studies using a murineVISTA-mIGg2a in vivo showed that therapeutic treatment as late as day 14had a beneficial effect on Clinical Disease Score in EAE. These ongoing,experiments look very exciting in that we may have identified a new axisin autoimmune disease intervention (FIG. 26). With this success we haveextended our studies using murine VISTA on a murine IgG1 or IgG2abackbone to exploit their cytophilic capacity. The Fc fusion constructsof VISTA in frame with the IgG1 Fc (both wild-type IgG1 and the existingnon-FcR-binding IgG1) have been produced. Each of these soluble VISTAmolecules was tested to determine if they can suppress EAE and it isshown that both VISTA-IgG1 and VISTA-IgG2a suppress the development andprogression of EAE (FIG. 27). While these early results suggest that adimeric, cytophilic VISTA will have activity in vivo, we will also beprepared to tetramerize using site specific biotinylation and complexingwith avidin for multimerization. The proposed studies leverage thisexpertise for the systematic generation and analysis of a set ofmultivalent reagents to modulate T cell function in vitro and, inparticular, in the context of EAE. Finally, we believe that the effortsdescribed in this proposal hold substantial promise for the developmentof new therapeutic strategies and will be of considerable benefit to theentire community interested in autoimmunity and T cell function ingeneral.

Example 26 VISTA-Ig Conjugate Reduces EAE Progression

Experimental Autoimmune Encephalomyelitis (EAE) is a model of multiplesclerosis. EAE was induced by immunizing mice with 175 μg MOG/CFA andpertussis toxin (PT) 300 ng (day 0, 2). On day 14, 17, and 20, 150 μgVISTA-IgG 2a (n=8) or 150 μg control IgG2a (n=8) was administered. Thedata is shown in FIG. 26 as the mean±SEM. In another experiment, on day6, mice were treated with 3 doses per week of 150 μg control IgG1 (n=3),150 μg control IgG2a (n=6), 150 μg mVISTA-IgG1 (n=3), or 150 μg mVISTAIgG2a (n=6) (two weeks in total). The data is shown in FIG. 27 as themean±SEM. In another experiment, on day 14, mice were treated with 3doses per week of PBS (n=6), 100 μg control IgG2a (n=6), 300 μg controlIgG2a (n=6), 100 μg VISTA-IgG2a (n=6), or 300 μg mVISTA IgG2a (n=6) (twoweeks in total). The data is shown in FIG. 28 as the mean±SEM. Thus, aVISTA-Ig fusion protein has a therapeutic effect on an inflammatorycondition, e.g., multiple sclerosis.

Example 27 Analysis of VISTA Expression in Human Cells and Suppressionby VISTA-Ig

The expression pattern of VISTA and its suppression by administration ofa VISTA-Ig fusion protein was examined in human cell samples.

Materials and Methods

Production of VISTA-Ig Fusion Protein—

A fusion protein was created consisting of amino acids 16-194 from theextracellular IgV domain of human VISTA and a form of human IgG1 mutatedfor low binding of Fc receptors. The VISTA sequence was cloned into theSpeI-BamHI sites of the vector CDM7B. Protein was produced by transienttransfection of Freestyle CHO cells using Freestyle transfection reagentand protein-free Freestyle Expression Media according to manufacturerinstructions (Invitrogen). Supernatant was harvested after 5 days ofgrowth and purified by protein G affinity columns. Protein wasconcentrated using 10K MWCO spin columns (Amicon).

Cell Preparation—

Human apheresis samples were obtained from unidentified healthy humandonors. For culture experiments, blood was layered onto Lymphoprep (PAA)and isolated by density-gradient centrifugation. Interface cells werewashed twice in PBS, then once in MACS buffer before undergoing magneticbead selection with Miltenyi CD4 Negative selection kit 11, CD8 NegativeSelection Kit, or the CD4 Memory T cell selection kit according tomanufacturer instructions. For effector cell isolation, CD4 T cells weresubsequently depleted of CD27⁺ cell types with Miltenyi CD27 positiveselection beads.

Culture—

T cells were plated at 2×10⁵ cells per well in 96-well flat-bottomplates coated with anti-CD3 (clone OKT3, BioXCell) and either VISTA-Igor control-Ig (ZZ, R&D biosystems). Unless otherwise indicated, anti-CD3was coated at 2.5 μg/ml mixed together with 10 μg/ml (ratio 1:4)VISTA-Ig or control-Ig protein in PBS at 4° C. overnight. Wells werewashed twice with complete media before adding cells. When indicated, atitrated amount of anti-CD28 (Miltenyi Biotech) was included in thecoating mix, or 50 ng/ml of IL-2, IL-4, IL-7 or IL-15 (Peprotech) wasadded to the culture media. Cultures were analyzed on day 2 for earlyactivation markers, and on day 5 for late activation markers or CFSEprofiles.

Flow Cytometry—

For staining following culture, cells were harvested and transferredinto V-bottomed 96-well plates. Cells were washed and stained in HBSS/5%BCS staining buffer containing antibodies (CD4, CD8, CD25, CD69, CD45RA;BD biosciences) and near-infrared fixable live-dead dye (Invitrogen).Cells were washed and fixed with BD fixation buffer before analysis.

For staining for VISTA expression, whole blood was washed and stainedwith PBA buffer (PBS/0.1% BSA/0.1% sodium azide) containing antibodiesfor extracellular markers. Antibodies against CD4, CD8, CD3, CD45RA,CD56, CD11b, CD11c, CD123, HLA-DR, CD14 and CD16 were purchased from BDbiosciences and anti-VISTA was produced as described herein. To stainFoxP3 intracellularly, Foxp3 Fixation/Permeabilization Concentrate andDiluent kit from eBiosciences and anti-FoxP3 antibody from BDbiosciences were used. See FIG. 33D.

Samples were acquired on a LSRII Fortessa (Becton & Dickinson, San Jose,Calif., USA) with FACSDiva software v6.1.2 (Becton & Dickinson) andanalysed with FlowJo software (Tree Star, Inc.). Graphs were createdusing graphed using Prism 5 (GraphPad Software, Inc.)

Results

The Human VISTA Protein—

A BLAST of the mouse VISTA sequence against the human genome identifieschromosome 10 open reading frame 54 (C10orf54 or platelet receptor Gi24precursor, GENE ID: 64115) with an e-value of 8e-165 and 77% identity.Common with mouse VISTA, this protein is predicted to encode a type Itransmembrane protein with a single extracellular IgV domain. HumanVISTA is a 311 amino acid (aa) long, consisting of a 32-aa signalpeptide, a 130-aa extracellular IgV domain, 33-aa stalk region, 20-aatransmembrane domain and a long 96-aa cytoplasmic tail. See amino acidsequence of SEQ ID NO: 16.

VISTA Expression Analysis—

The expression of VISTA healthy human tissues was examined by real-timePCR analysis of a cDNA tissue panel (Origene) FIG. 29A) Similar to mousetissues, VISTA was predominantly expressed in haematopoietic tissues orin tissues that contain significant numbers of haematopoietic tissues.This is consistent with importance of VISTA in immune related functions.Interestingly, expression of VISTA was particularly high in humanplacenta, which may be indicative of a functional role for VISTA inmaintenance of tolerance to the allogeneic environment of pregnancy.This pattern of expression was found to follow a similar trend to thatof VISTA's closest homologue PD-L1 (FIG. 29B).

Next, VISTA protein expression was examined within the haematopoieticcompartment by flow cytometry. PBMCs were isolated from peripheral bloodand stained with the anti-VISTA monoclonal antibody GA1. VISTA washighly expressed by the majority of monocytes, dendritic cells and byapproximately 20% of CD4 and CD8 T cells (FIG. 30). VISTA expression wasobserved within both of the ‘patrolling’ (CD14^(dim)CD16⁺) and‘inflammatory’ (CD14⁺CD16^(+/−)) subsets of blood monocytes, and withinboth lymphoid and myeloid subsets of dendritic cell.

Functional Effect of VISTA on T Cell Function—

VISTA has previously been demonstrated to have a negative impact onmouse T cell immune responses (Wang, et al. (2011) J. Exp. Med. pages1-16). Whether VISTA had the same role in the human cell-mediated immuneresponse was examined. An Ig fusion protein was created, consisting ofthe extracellular domain of VISTA and the Fc region of human IgGcontaining mutations for reduced Fc receptor binding. 10 μg/ml ofVISTA-Ig or control Ig was immobilized on plates along with 2.5 μg/ml ofanti-CD3 (OKT3) and then proliferation was measured by CFSE dilution.VISTA was found to suppress CFSE dilution of bulk purified CD4 (FIG.31A) and CD8 (FIG. 31B) T cells. The suppression by VISTA is comparableto that induced by PD-L1-Ig (R&D biosystems). Additionally, VISTA-Ig waseffective at suppression of memory (CD45RO⁺, FIG. 31C) and effector(CD27⁻, FIG. 31D) subsets. Comparison of mouse VISTA and human VISTA onhuman CD4 T cells demonstrated that VISTA is cross-reactive acrossspecies. Titration of human VISTA-Ig and human VISTA-Ig over differentconcentrations of OKT3, showed that higher concentrations of OKT3 can beovercome by higher concentrations of VISTA (FIGS. 32A and 32B).

To gain some insight into the mechanism of suppression, the status ofcells was examined following activation in the presence or absence ofVISTA-Ig. During 2 days of culture, upregulation by anti-CD3 of theearly activation markers CD25 and CD69 was blocked by VISTA-Ig (FIGS.33A & 33B) Similarly, after 5 days of culture, the shift from expressionof CD45RA to CD45RO, indicative of antigen-experience was prevented(FIG. 33C). VISTA had no affect on cell viability. Consistent with ablock in proliferation, cells treated with VISTA-Ig had forward andside-scatter profiles similar to unstimulated cells rather than blastingcells seen with OKT3 alone. To determine if the suppression induced byVISTA is stable, cells were cultured on anti-CD3 and VISTA-Ig for twodays, and then moved onto anti-CD3 alone for 3 days. This furtherstimulation was unable to rescue suppression as shown in FIGS. 34A and34B.

Next, the effect of VISTA-Ig on cytokine production was examined. Cellswere stimulated with plate-bound OKT3 for 5 days in the presence ofincreasing amounts of VISTA-Ig, and then the concentration of variouscytokines was measured in culture supernatants by cytometric bead array.Only trace levels of IL-2, IL-4 or IL-6 were detected (<5 pg/ml) and nodifferences were observed. However, VISTA-Ig significantly reducedproduction of IL-10, TNFα and IFNγ by CD4 (FIG. 35A) and CD8 (FIG. 35B)T cells, and there was a trend towards a modest decrease in IL-17production.

Factors that were able to overcome the VISTA-induced suppression of Tcells were also examined. Anti-CD28 agonistic antibody provides potentcostimulation to T cells, and so titred into the cultures to challengeVISTA suppression (FIG. 36A-C). Although lower amounts of anti-CD28 wereunable to overcome VISTA, when anti-CD28 was included at a coatingconcentration of 1 μg/ml VISTA was unable to block proliferation.Similarly, while low concentrations of VISTA could be overcome by theaddition of cytokines such as IL-2, IL-7 and IL-15, higherconcentrations of VISTA were still suppressive even with aphysiologically high concentration of cytokine at 50 ng/ml.

Thus, VISTA-Ig fusion protein may be used as a negative regulator ofinflammation because it may significantly reduce production of IL-10,TNFα and IFNγ by CD4 and CD8 T cells. This, in turn, may lead to atherapeutic downregulation of the immune response and provide relieffrom autoimmune or inflammatory disorders.

Example 28 VISTA Expression on Tumour Infiltrating Leukocytes (TILs) inHuman Colorectal Carcinoma and Relationship to Disease Stage andPrognosis

We previously demonstrated that murine TILs express very high levels ofVISTA, and blocking antibody to VISTA reduces tumour growth (2). We havealso demonstrated VISTA expression in both peripheral blood mononuclearcells (PBMCs) and healthy colonic lamina propria mononuclear cells(LPMCs) in humans and hypothesize that VISTA is expressed ontumour-infiltrating leukocytes in human colorectal carcinoma (CRC). Thisexample describes characterization of VISTA expression in CRC, adjacent“healthy” mucosa and paired peripheral blood by immunofluorescencemicroscopy and flow cytometry. Tissue sections give valuable informationabout the architecture of VISTA expression within the TME. Flowcytometry allows more extensive characterization of VISTA-expressing andnon-expressing cells, including the frequency and activation status ofTILs such as myeloid-derived suppressor cells (MDSCs), tumour-associatedmacrophages (TAMs), dendritic cells (DCs) and regulatory T cells(Tregs). VISTA expression in CRC is also related to clinical andpathological data to demonstrate the association between VISTAexpression and prognostic markers, such as tumour stage.

Antibody-Mediated VISTA Blockade Inhibits Tumour Growth:

Previous studies in the lab have established that VISTA is a potentimmune suppressive ligand that binds to an unknown receptor on T cellsindependently of PD-1 (1). VISTA suppresses T-cell proliferation andcytokine production when expressed on APCs. VISTA overexpression ontumour cells impaired protective anti-tumour immunity in vaccinatedhosts. Anti-VISTA clone 13F3 functionally blocked the suppressiveactivity of VISTA in vitro, and exacerbated disease progression ofExperimental Autoimmune Encephalomyelitis (EAE) (75). Furthermore, 13F3administration significantly reduced tumour growth in multipletransplantable tumour systems including a bladder tumour MB49, aMethylcholanthrene (MCA)-105 fibrosarcoma, a thymoma EG7, an ovariantumour ID8 (FIG. 37), and B16F10 melanoma. In addition, we haveconfirmed that aVISTA-mediated tumour rejection is correlated withenhanced regional anti-tumour T-cell response, as measured by IFNgELISPOT from MB49 tumour-draining lymph node (LN) lymphocytes.

To further demonstrate the direct translational relevance of the abovemurine data to the pathogenesis of cancer in man, our group hasinitiated a multi-site, NIHR-funded, CLRN-registered observational studyinvestigating defects in mucosal immunology in inflammatory boweldisease and CRC (REC 10/H0804/65, NIHR CRN 9929). Next, we perfected theextraction of LPMCs from intestinal resection specimens, for use inmulti-colour flow cytometry and functional assays. Using anti-VISTAclones GA1 and HCl (APS Biotech Ltd), we subsequently identified VISTAexpression in human peripheral blood and lamina propria monocytes andLin-HLA-DR+ DCs and monocytes, which has not previously been described(FIG. 38). Consequently, in addition to being expressed in healthyLPMCs, we hypothesise that VISTA is expressed in TILs in adjacent CRC.

We have optimised 8-10 colour Fluorescence Assisted Cell Sorting (FACS)antibody panels to determine the frequency and activation status ofimmune cells, in addition to cytokine and transcription factorexpression, and applied these panels to PBMCs and LPMCs. FACS is thenused to distinguish populations of TILs in the colon tumour samplesbased on population-specific surface markers. For example human MDSCsare found to be CD11b+, CD33+, HLA-DR- and are further divided intoCD14+ and CD14− subsets. The VISTA positive and negative sub-populationsof these are further distinguished with the help of the 2 antibodyclones our lab has previously developed for the molecule (FIG. 41; GA1and HCl). Activation markers like CD69 on the T cells, CD64, CD62L ontumour associated macrophages, etc. are compared between the VISTApositive and negative subpopulations. These can then be further studied,as in Example 29, in in vitro conditions.

Sections of tumour samples are frozen in OCT, then analysed for VISTAexpression. These are be stained by immunofluorescence, or byimmunohistochemistry with haematoxylin counterstain. Immunofluorescencestaining will use anti-VISTA antibodies along with antibodies specificfor other cells in the TME (e.g. epithelial cells, MDSCs, tolerogenicDCs, T cells, B cells and other immune cells. This demonstrates themicro-anatomical localisation of VISTA-expressing cells and theirspatial interaction with other cells in the TME. Immunohistochemistrywith a haematoxylin counterstain elucidates the pathology and morphologyof the tumour, and how VISTA is associated with different pathology(e.g. inflamed or necrotic regions).

Further, VISTA expression is compared to clinical and pathological datasuch as Duke's stage, pathological TNM staging and histological featuressuch as neural invasion and degree of differentiation, to determine howVISTA expression relates to proxies for clinical outcome. Using theparadigm of COX-2 expression in CRC (“High” COX-2 expression in Duke's A66% vs. Duke's D 100%), a sample size of 80 (n=20 in each Duke's staginggroup) has an 84% power (α=0.05) to detect a 33% difference between anytwo groups (76).

By classifying the samples in a globally and clinically accepted manner,the study provides clinically relatable data. Characterization of theTILs in patient samples will outline which cells express VISTA in humansand how expression within each subset changes with each stage of cancerdevelopment and is associated with outcome. For example, it has beenfound that in human ovarian cancer, large infiltration of FoxP3+ Tregsis associated with a poorer prognosis. This aim will also drive furtherin vitro studies, as in Example 29.

Example 29 The Functional Role of VISTA Expression on TumourInfiltrating Leukocytes (TIL)

Our murine data suggest that VISTA-positive Tregs are more suppressivethan VISTA-negative Tregs. Consequently, we hypothesized thatVISTA-expressing TILs will be more suppressive on effector immuneresponses than VISTA-negative TILs. This hypothesis is tested using twocomplementary methods. First, TAMs, MDSCs, DCs and Tregs from coloniccancer and healthy control samples are sorted for VISTA positive versusnegative subsets. The suppressive or stimulatory nature of these cellstowards T cells is determined in vitro, followed by further mechanisticexperiments. Second, VISTA expression is knocked down by retroviral RNAito determine how this influences the suppressive nature of different TILcell types in vitro. This approach helps further our understanding ofhow potential treatments may impact the TME.

We have demonstrated that VISTA profoundly suppresses CD4+ and CD8+ Tcell responses (FIG. 39). Specifically, FIG. 3 shows the proliferationsuppression in an in vitro assay using plate-bound VISTA-Ig in an aCD3proliferation assay. This example further examines how VISTA influencesthe function of different cell types within the TME. Understanding thefunctional role of VISTA in colon cancer further provides the basis foranticancer treatment made possible by the use of anti-VISTA antibodies.

Fluorescence Assisted Cell Sorting is used to obtain individualpopulations of myeloid or lymphoid derived TILs (tumour associatedmacrophages, myeloid derived suppressor cells, T cells, B cells, andDC's) from tumour samples based on cell-specific and activation markers.They are then transfected with lentiviruses expressing iRNA to knockdownVISTA or PD-L1 expression, or retroviruses expressing high-level VISTA,PD-L1 or ‘empty’. PD-L1 will be used as a positive control and to givedata context within the B7-CD28 family. Constructs for four unique 29mershRNAs for hVISTA and a control non-functional 29mer scrambled shRNA ina retroviral vector are commercially available (Origene, Rockville,Md.). These are guaranteed to achieve greater than 70% knockdown.Following transfection, TILs are tested for suppressive/stimulatoryability in standard mixed lymphocyte reaction (MLR) cultures with CFSElabelled responder T cells. Cytokine production is also measured todetermine how VISTA impacts differentiation.

Example 30 Screening a Panel of Anti-VISTA Antibodies and ComparingActivity with Existing Anti-PD-L1 mAb and Identification of VISTABlocking Antibodies

Anti-VISTA antibodies previously produced in our lab have proved to beeffective in diminishing tumour growth in mice, and closest homologue toVISTA, PD-L1 is already performing well in clinical trials for treatmentof melanoma. This example describes testing a panel of anti-VISTAantibodies for efficacy in enhancing T cell proliferation in vitro,compared to the efficacy of either anti-PD-L1 alone, or the combinationof VISTA and PD-L1 blockade.

The role of immune checkpoint pathways such as PD-L1/PD-1 and CTLA-4 hasbeen well documented in human patients, and CTLA-4 blockade (Ipilumimab)the first immunological treatment with any efficacy against late-stagemelanoma (77). The use of anti-VISTA treatment in combination withblockade of another immunological checkpoint protein may enhanceanti-tumour responses still further. Our lab tested this theory usingthe cell line B16F10 in C57BL/6 mice. Due to its poor immunogenicity,B16F10 also represents a very challenging murine tumour system forimmune-interventions against cancer. Prophylactic treatment (day-2) withaVISTA halted B16F10 tumour progression significantly, whereas blockadeof another immune checkpoint protein, PD-L1, failed to have any impact(FIG. 40). When aVISTA was administered therapeutically (day+4),efficacy as a single reagent was not detected. However, an additiveimpact on tumour growth was seen when VISTA blockade was combined withPD-L1 blockade (FIG. 40). Greater synergistic effect was observed invitro when VISTA-Ig and PD-L1-Ig were used together to suppress T cellactivation.

Our results lead us to the belief that VISTA synergizes with PD-L1 tomaximally suppress T-cell responses. In this example, we test a panel ofanti-VISTA antibodies for efficacy in enhancing T cell proliferation invitro, and contrast this with either anti-PD-L1 alone, or thecombination of VISTA and PD-L1 blockade.

We have previously generated clones of anti-VISTA, GA1 and HCl. Stainingwith GA1 mAb is shown in FIG. 41. GA1 was selected by its ability tobind hVISTA-Ig but not Ig in an ELISA and then selected by specificstaining of GFP-VISTA transduced cells but not control transduced cells.As shown (FIG. 41) using mAb supernatant, GA1 binds over 50% ofmonocytes in peripheral blood and a small percentage of lymphocytes, allwhich are blocked by VISTA-Ig. A more extensive panel of anti-humanVISTA antibodies is generated and tested for efficacy in blocking theVISTA pathway. To generate further antibodies, we will mice with anirradiated VISTA-expressing EL4 cell line, followed by monthly boostswith VISTA-Ig. Titres are confirmed by ELISA using VISTA-Ig againsthuman IgG to control for responses against the Fc region. Spleens arethen used to generate myelomas. Supernatants are then screened forbinders using human PBMCs and also VISTA-expressing cell lines. Positiveclones are sub-cloned and cryopreserved. Based on initial data, fromeach fusion, we expect 5-10 candidate binders, and will thereforeimmunize 10 mice in the first instance.

Antibodies are tested for efficacy in blocking T cell responses inducedby VISTA expressing APCs. Human apheresis leukocyte-enriched cones(e.g., available from King's College Hospital). From each sample, wetypically can obtain about 10⁹ PBMCs. Large numbers of CD4 T cells, andfrom mismatched donors CD14+ monocytes, are both isolated by Miltenyibead selection and cryopreserved. This gives a relatively consistentcell population to screen antibodies with in mixed lymphocyte reactions.Human blood monocytes express high levels of VISTA (FIG. 41), andtherefore are a suitable stimulatory population to use. Proliferativeresponses of T cells are measured by CFSE dilution in the presence ofanti-vista antibodies relative to control Ig. Additionally, anti-PD-L1antibody is also used, as it is a well-characterized agent useful as apositive control and for comparison. We also test the effect of usingboth reagents together for synergy, and envision using the two reagentstogether as treatment.

Using similar methods, anti-VISTA antibodies are tested for efficacy inblocking T cell responses induced by the VISTA-expressing APC cell linessuch as K-562.

Additionally, anti-VISTA antibodies are tested for the ability to blockVISTA-Ig suppression of anti-CD3 stimulation. As described in Example 3above, VISTA (PD-L3)-Ig suppressed the proliferation of bulk purifiedCD4+ and CD8+ T cells in response to plate-bound anti-CD3 stimulation,as determined by arrested cell division (see FIG. 9A-B).

VISTA-inhibitory antibodies identified in these assays are furthertested in vivo for promoting anti-tumor responses in animal models ofcancer, e.g., as further described in the preceding examples.Additionally, antibodies that show efficacy may be humanized and furtherdeveloped for potential therapeutic use.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

REFERENCES

-   1. Wang, L., et al., VISTA, a novel Ig-superfamily ligand that    negatively regulates T cell responses. J. Exp. Med., 2011.    208(3): p. 577-92.-   2. Wang, L., et al., Programmed death 1 ligand signaling regulates    the generation of adaptive Foxp3+CD4+ regulatory T cells. Proc Natl    Acad Sci USA, 2008. 105(27): p. 9331-6.-   3. Hodi, F. S., Overcoming immunological tolerance to melanoma:    Targeting CTLA-4. Asia Pac J Clin Oncol, 2010. 6 Suppl 1: p. S16-23.-   4. Tarhini, A., E. Lo, and D. R. Minor, Releasing the brake on the    immune system: ipilimumab in melanoma and other tumors. Cancer    Biother Radiopharm, 2010. 25(6): p. 601-13.-   5. Kaehler, K. C., et al., Update on immunologic therapy with    anti-CTLA-4 antibodies in melanoma: identification of clinical and    biological response patterns, immune-related adverse events, and    their management. Semin Oncol, 2010. 37(5): p. 485-98.-   6. Wing, K., et al., CTLA-4 control over Foxp3+ regulatory T cell    function. Science, 2008. 322(5899): p. 271-5.-   7. Chambers, C. A., T. J. Sullivan, and J. P. Allison,    Lymphoproliferation in CTLA-4-deficient mice is mediated by    costimulation-dependent activation of CD4+ T cells. Immunity, 1997.    7(6): p. 885-95.-   8. Waterhouse, P., et al., Lymphoproliferative disorders with early    lethality in mice deficient in Ctla-4. Science, 1995. 270(5238): p.    985-8.-   9. Tivol, E. A., et al., Loss of CTLA-4 leads to massive    lymphoproliferation and fatal multiorgan tissue destruction,    revealing a critical negative regulatory role of CTLA-4.    Immunity, 1995. 3(5): p. 541-7.-   10. Zheng, S. G., et al., TGF-beta requires CTLA-4 early after T    cell activation to induce FoxP3 and generate adaptive CD4+CD25+    regulatory cells. J Immunol, 2006. 176(6): p. 3321-9.-   11. van Elsas, A., A. A. Hurwitz, and J. P. Allison, Combination    immunotherapy of B16 melanoma using anticytotoxic T    lymphocyte-associated antigen 4 (CTLA-4) and granulocyte/macrophage    colony-stimulating factor (GM-CSF)-producing vaccines induces    rejection of subcutaneous and metastatic tumors accompanied by    autoimmune depigmentation. J Exp Med, 1999. 190(3): p. 355-66.-   12. Hoos, A., et al., Development of ipilimumab: contribution to a    new paradigm for cancer immunotherapy. Semin Oncol, 2010. 37(5): p.    533-46.-   13. Calabro, L., et al., Clinical studies with anti-CTLA-4    antibodies in non-melanoma indications. Semin Oncol, 2010. 37(5): p.    460-7.-   14. Attia, P., et al., Autoimmunity correlates with tumor regression    in patients with metastatic melanoma treated with anti-cytotoxic    T-lymphocyte antigen-4. J Clin Oncol, 2005. 23(25): p. 6043-53.-   15. Freeman, G. J., et al., Engagement of the PD-1 immunoinhibitory    receptor by a novel B7 family member leads to negative regulation of    lymphocyte activation. J Exp Med, 2000. 192(7): p. 1027-34.-   16. Butte, M. J., et al., Programmed death-1 ligand 1 interacts    specifically with the b7-1 costimulatory molecule to inhibit T cell    responses. Immunity, 2007. 27(1): p. 111-22.-   17. Nishimura, H., et al., Development of lupus-like autoimmune    diseases by disruption of the PD-1 gene encoding an ITIM    motif-carrying immunoreceptor. Immunity, 1999. 11(2): p. 141-51.-   18. Nishimura, H., et al., Autoimmune dilated cardiomyopathy in PD-1    receptor-deficient mice. Science, 2001. 291(5502): p. 319-22.-   19. Keir, M. E., et al., PD-1 and its ligands in tolerance and    immunity Annu Rev Immunol, 2008. 26: p. 677-704.-   20. Gao, Q., et al., Overexpression of PD-L1 significantly    associates with tumor aggressiveness and postoperative recurrence in    human hepatocellular carcinoma. Clin Cancer Res, 2009. 15(3): p.    971-9.-   21. Dong, H. and L. Chen, B7-H1 pathway and its role in the evasion    of tumor immunity J Mol Med, 2003. 81(5): p. 281-7.-   22. Zou, W. and L. Chen, Inhibitory B7-family molecules in the    tumour microenvironment. Nat Rev Immunol, 2008. 8(6): p. 467-77.-   23. Dong, H., et al., Tumor-associated B7-H1 promotes T-cell    apoptosis: a potential mechanism of immune evasion. Nat Med, 2002.    8(8): p. 793-800.-   24. Blank, C., et al., PD-L1/B7H-1 inhibits the effector phase of    tumor rejection by T cell receptor (TCR) transgenic CD8+ T cells.    Cancer Res, 2004. 64(3): p. 1140-5.-   25. Blank, C., T. F. Gajewski, and A. Mackensen, Interaction of    PD-L1 on tumor cells with PD-1 on tumorspecific T cells as a    mechanism of immune evasion: implications for tumor immunotherapy.    Cancer Immunol Immunother, 2005. 54(4): p. 307-14.-   26. Iwai, Y., et al., Involvement of PD-L1 on tumor cells in the    escape from host immune system and tumor immunotherapy by PD-L1    blockade. Proc Natl Acad Sci USA, 2002. 99(19): p. 12293-7.-   27. Geng, H., et al., HSP70 vaccine in combination with gene therapy    with plasmid DNA encoding sPD-1 overcomes immune resistance and    suppresses the progression of pulmonary metastatic melanoma. Int J    Cancer, 2006. 118(11): p. 2657-64.-   28. Hirano, F., et al., Blockade of B7-H1 and PD-1 by monoclonal    antibodies potentiates cancer therapeutic immunity Cancer Res, 2005.    65(3): p. 1089-96.-   29. Curiel, T. J., et al., Blockade of B7-H1 improves myeloid    dendritic cell-mediated antitumor immunity Nat Med, 2003. 9(5): p.    562-7.-   30. Brahmer, J. R., et al., Phase I study of single-agent    anti-programmed death-1 (MDX-1106) in refractory solid tumors:    safety, clinical activity, pharmacodynamics, and immunologic    correlates. J Clin Oncol, 2010. 28(19): p. 3167-75.-   31. Sica, G. L., et al., B7-H4, a molecule of the B7 family,    negatively regulates T cell immunity Immunity, 2003. 18(6): p.    849-61.-   32. Prasad, D. V., et al., B7S1, a novel B7 family member that    negatively regulates T cell activation. Immunity, 2003. 18(6): p.    863-73.-   33. Yi, K. H. and L. Chen, Fine tuning the immune response through    B7-H3 and B7-H4. Immunol Rev, 2009. 229(1): p. 145-51.-   34. Kryczek, I., et al., B7-H4 expression identifies a novel    suppressive macrophage population in human ovarian carcinoma. J Exp    Med, 2006. 203(4): p. 871-81.-   35. Kryczek, I., et al., Cutting edge: induction of B7-H4 on APCs    through IL-10: novel suppressive mode for regulatory T cells. J    Immunol, 2006. 177(1): p. 40-4.-   36. Shevach, E. M., CD4+CD25+ suppressor T cells: more questions    than answers. Nat Rev Immunol, 2002. 2(6): p. 389-400.-   37. Nishikawa, H. and S. Sakaguchi, Regulatory T cells in tumor    immunity Int J Cancer, 2010. 127(4): p. 759-67.-   38. Yamaguchi, T. and S. Sakaguchi, Regulatory T cells in immune    surveillance and treatment of cancer. Semin Cancer Biol, 2006.    16(2): p. 115-23.-   39. Curiel, T. J., et al., Specific recruitment of regulatory T    cells in ovarian carcinoma fosters immune privilege and predicts    reduced survival. Nat Med, 2004. 10(9): p. 942-9.-   40. Zou, W., Regulatory T cells, tumour immunity and immunotherapy.    Nat Rev Immunol, 2006. 6(4): p. 295-307.-   41. Sharma, M. D., et al., Plasmacytoid dendritic cells from mouse    tumor-draining lymph nodes directly activate mature Tregs via    indoleamine 2,3-dioxygenase. J Clin Invest, 2007. 117(9): p.    2570-82.-   42. Tacke, F. and G. J. Randolph, Migratory fate and differentiation    of blood monocyte subsets. Immunobiology, 2006. 211(6-8): p. 609-18.-   43. Shortman, K. and S. H. Naik, Steady-state and inflammatory    dendritic-cell development. Nat Rev Immunol, 2007. 7(1): p. 19-30.-   44. Leon, B. and C. Ardavin, Monocyte-derived dendritic cells in    innate and adaptive immunity Immunol Cell Biol, 2008. 86(4): p.    320-4.-   45. Auffray, C., M. H. Sieweke, and F. Geissmann, Blood monocytes:    development, heterogeneity, and relationship with dendritic cells.    Annu Rev Immunol, 2009. 27: p. 669-92.-   46. Geissmann, F., et al., Development of monocytes, macrophages,    and dendritic cells. Science, 2010. 327(5966): p. 656-61.-   47. Qu, C., et al., Role of CCR8 and other chemokine pathways in the    migration of monocyte-derived dendritic cells to lymph nodes. J Exp    Med, 2004. 200(10): p. 1231-41.-   48. Geissmann, F., S. Jung, and D. R. Littman, Blood monocytes    consist of two principal subsets with distinct migratory properties.    Immunity, 2003. 19(1): p. 71-82.-   49. Sunderkotter, C., et al., Subpopulations of mouse blood    monocytes differ in maturation stage and inflammatory response. J    Immunol, 2004. 172(7): p. 4410-7.-   50. Randolph, G. J., et al., Differentiation of phagocytic monocytes    into lymph node dendritic cells in vivo Immunity, 1999. 11(6): p.    753-61.-   51. Robben, P. M., et al., Recruitment of Gr-1+ monocytes is    essential for control of acute toxoplasmosis. J Exp Med, 2005.    201(11): p. 1761-9.-   52. Serbina, N. V., et al., TNF/iNOS-producing dendritic cells    mediate innate immune defense against bacterial infection.    Immunity, 2003. 19(1): p. 59-70.-   53. Copin, R., et al., MyD88-dependent activation of    B220-CD11b+LY-6C+ dendritic cells during Brucella melitensis    infection. J Immunol, 2007. 178(8): p. 5182-91.-   54. Geissmann, F., et al., Blood monocytes: distinct subsets, how    they relate to dendritic cells, and their possible roles in the    regulation of T-cell responses. Immunol Cell Biol, 2008. 86(5): p.    398-408.-   55. Krutzik, S. R., et al., TLR activation triggers the rapid    differentiation of monocytes into macrophages and dendritic cells.    Nat Med, 2005. 11(6): p. 653-60.-   56. Leon, B., M. Lopez-Bravo, and C. Ardavin, Monocyte-derived    dendritic cells formed at the infection site control the induction    of protective T helper 1 responses against Leishmania.    Immunity, 2007. 26(4): p. 519-31.-   57. Le Borgne, M., et al., Dendritic cells rapidly recruited into    epithelial tissues via CCR6/CCL20 are responsible for CD8+ T cell    crosspriming in vivo. Immunity, 2006. 24(2): p. 191-201.-   58. Nakano, H., et al., Blood-derived inflammatory dendritic cells    in lymph nodes stimulate acute T helper type 1 immune responses. Nat    Immunol, 2009. 10(4): p. 394-402.-   59. Rabinovich, G. A., D. Gabrilovich, and E. M. Sotomayor,    Immunosuppressive strategies that are mediated by tumor cells. Annu    Rev Immunol, 2007. 25: p. 267-96.-   60. Gabrilovich, D. I. and S. Nagaraj, Myeloid-derived suppressor    cells as regulators of the immune system. Nat Rev Immunol, 2009.    9(3): p. 162-74.-   61. Wilcox, R. A., Cancer-associated myeloproliferation: old    association, new therapeutic target. Mayo Clin Proc, 2010. 85(7): p.    656-63.-   62. Ostrand-Rosenberg, S. and P. Sinha, Myeloid-derived suppressor    cells: linking inflammation and cancer. J Immunol, 2009. 182(8): p.    4499-506.-   63. Mango, I., et al., Tumor-induced tolerance and immune    suppression by myeloid derived suppressor cells Immunol Rev, 2008.    222: p. 162-79.-   64. Corzo, C. A., et al., HIF-1alpha regulates function and    differentiation of myeloid-derived suppressor cells in the tumor    microenvironment. J Exp Med, 2010. 207(11): p. 2439-53.-   65. Gabrilovich, D., Mechanisms and functional significance of    tumour-induced dendritic-cell defects. Nat Rev Immunol, 2004.    4(12): p. 941-52.-   66. Melief, C. J., Cancer immunotherapy by dendritic cells.    Immunity, 2008. 29(3): p. 372-83.-   67. Steinman, R. M., D. Hawiger, and M. C. Nussenzweig, Tolerogenic    dendritic cells. Annu Rev Immunol, 2003. 21: p. 685-711.-   68. Ghiringhelli, F., et al., Tumor cells convert immature myeloid    dendritic cells into TGF-beta-secreting cells inducing CD4+CD25+    regulatory T cell proliferation. J Exp Med, 2005. 202(7): p. 919-29.-   69. Conejo-Garcia, J. R., et al., Tumor-infiltrating dendritic cell    precursors recruited by a beta-defensin contribute to vasculogenesis    under the influence of Vegf-A. Nat Med, 2004. 10(9): p. 950-8.-   70. Huarte, E., et al., Depletion of dendritic cells delays ovarian    cancer progression by boosting antitumor immunity Cancer Res, 2008.    68(18): p. 7684-91.-   71. Cubillos-Ruiz, J. R., et al., Polyethylenimine-based siRNA    nanocomplexes reprogram tumor-associated dendritic cells via TLR5 to    elicit therapeutic antitumor immunity J Clin Invest, 2009.    119(8): p. 2231-44.-   72. Bak, S. P., et al., Murine ovarian cancer vascular leukocytes    require arginase-1 activity for T cell suppression. Mol    Immunol, 2008. 46(2): p. 258-68.-   73. Scarlett, U. K., et al., In situ stimulation of CD40 and    Toll-like receptor 3 transforms ovarian cancerinfiltrating dendritic    cells from immunosuppressive to immunostimulatory cells. Cancer    Res, 2009. 69(18): p. 7329-37.-   74. Nesbeth, Y. C., et al., CD4+ T cells elicit host immune    responses to MHC class II-negative ovarian cancer through CCL5    secretion and CD40-mediated licensing of dendritic cells. J    Immunol, 2010. 184(10): p. 5654-62.-   75. Wang, L., et al., VISTA, a novel mouse Ig-superfamily ligand    that negatively regulates T cell responses. J Exp Med, 2010.-   76. Sheehan, K., K. Sheehan, and D. O'Donoghue, The relationship    between cyclooxygenase-2 expression and colorectal cancer.    JAMA, 1999. 282: p. 1254-7.-   77. Weber, J., Immune checkpoint proteins: a new therapeutic    paradigm for cancer—preclinical background: CTLA-4 and PD-1    blockade. Semin Oncol, 2010. 37(5): p. 430-9.

What is claimed is:
 1. A method for effecting a synergistic enhancementin CD8⁺ T cell immunity in a subject in need thereof comprisingadministering a synergistic combination comprising an antagonisticanti-VISTA antibody and a PD-1 antagonist, with the proviso that thecondition to which CD8⁺ T cell immunity is enhanced does not comprisecancer.
 2. The method of claim 1, wherein the PD-1 antagonist is anantibody.
 3. The method of claim 2, wherein the PD-1 antagonist is ananti-PD-1 or anti-PD-L1 antibody.
 4. The method of claim 1 wherein theanti-VISTA antibody and the PD-1 antagonist may be in the same ordifferent compositions.
 5. The method of claim 1, wherein thesynergistic combination consists of an anti-VISTA antibody and a PD-1antagonist.
 6. The method of claim 5, wherein the PD-1 antagonistconsists of an anti-PD-1 antibody.
 7. The method of claim 1, wherein thesubject has an infection and the method results in a synergisticenhancement in CTL immunity against said infection.
 8. The method ofclaim 7, wherein the infection is caused by a virus.